Skin-infiltrating T cells and cytokine expression in Icelandic horses affected with insect bite hypersensitivity: a possible role for regulatory T cells

Equine insect bite hypersensitivity (IBH) is a seasonally recurrent, pruritic skin disorder caused by an IgE-mediated reaction to salivary proteins of biting flies, predominantly of the genus Culicoides. The aim of this study was to define T cell subsets and cytokine profile in the skin of IBH-affected Icelandic horses with particular focus on the balance between T helper (Th) 1, Th2 and T regulatory (Treg) cells. Distribution and number of CD4+, CD8+ and Forkhead box P3 (FoxP3)+ T cells were characterized by immunohistochemical staining in lesional and non-lesional skin of moderately and severely IBH-affected horses (n=14) and in the skin of healthy control horses (n=10). Using real-time quantitative reverse transcription-polymerase chain reaction, mRNA expression levels of Th2 cytokines (Interleukin (IL)-4, IL-5, IL-13), Th1 cytokines (Interferon-gamma), regulatory cytokines (Transforming Growth Factor beta1, IL-10) and the Treg transcription factor FoxP3 were measured in skin and blood samples. Furthermore, Culicoides nubeculosus specific serum IgE levels were assessed. Lesions of IBH-affected horses contained significantly higher numbers of CD4+ cells than skin of healthy control horses. Furthermore, the total number of T cells (CD4+ and CD8+) was significantly increased in lesional compared to non-lesional skin and there was a tendency (p=0.07) for higher numbers of CD4+ cells in lesional compared to non-lesional skin. While the number of FoxP3+ T cells did not differ significantly between the groups, the ratio of Foxp3 to CD4+ cells was significantly lower in lesions of severely IBH-affected horses than in moderately affected or control horses. Interestingly, differences in FoxP3 expression were more striking at the mRNA level. FoxP3 mRNA levels were significantly reduced in lesional skin, compared both to non-lesional and to healthy skin and were also significantly lower in non-lesional compared to healthy skin. Expression levels of IL-13, but not IL-4 or IL-5, were significantly elevated in lesional and non-lesional skin of IBH-affected horses. IL-10 levels were lower in lesional compared to non-lesional skin (p=0.06) and also lower (p=0.06) in the blood of IBH-affected than of healthy horses. No significant changes were observed regarding blood expression levels of Th1 and Th2 cytokines or FoxP3. Finally, IBH-affected horses had significantly higher Culicoides nubeculosus specific serum IgE levels than control horses. The presented data suggest that an imbalance between Th2 and Treg cells is a characteristic feature in IBH. Treatment strategies for IBH should thus aim at restoring the balance between Th2 and Treg cells.

Pregnancy induces numerical and functional changes of CD4+CD25 high regulatory T cells in patients with rheumatoid arthritis

OBJECTIVE: In a prospective study we investigated whether numerical and functional changes of CD4+CD25(high) regulatory T cells (Treg) were associated with changes of disease activity observed during pregnancy and post partum in patients with rheumatoid arthritis (RA). METHODS: The frequency of CD4+CD25(high) T cells was determined by flow cytometry in 12 patients with RA and 14 healthy women during and after pregnancy. Fluorescence-activated cell sorting (FACS) was used to sort CD4+CD25(high) T cells and CD4+CD25- T cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies alone or in co-culture to investigate proliferation and cytokine secretion. RESULTS: Frequencies of CD4+CD25(high) Treg were significantly higher in the third trimester compared to 8 weeks post partum in patients and controls. Numbers of CD4+CD25(high) Treg inversely correlated with disease activity in the third trimester and post partum. In co-culture experiments significantly higher amounts of IL10 and lowered levels of tumour necrosis factor (TNF)alpha and interferon (IFN)gamma were found in supernatants of the third trimester compared to postpartum samples. These findings were independent from health or disease in pregnancy, however postpartum TNFalpha and IFN gamma levels were higher in patients with disease flares. CONCLUSION: The amelioration of disease activity in the third trimester corresponded to the increased number of Treg that induced a pronounced anti-inflammatory cytokine milieu. The pregnancy related quantitative and qualitative changes of Treg suggest a beneficial effect of Treg on disease activity.

Prednisolone treatment induces tolerogenic dendritic cells and a regulatory milieu in myasthenia gravis patients

FOXP3-expressing naturally occurring CD4(+)CD25(high) T regulatory cells (Treg) are relevant in the control of autoimmunity, and a defect in this cell population has been observed in several human autoimmune diseases. We hypothesized that altered functions of peripheral Treg cells might play a role in the immunopathogenesis of myasthenia gravis, a T cell-dependent autoimmune disease characterized by the presence of pathogenic autoantibodies specific for the nicotinic acetylcholine receptor. We report in this study a significant decrease in the in vitro suppressive function of peripheral Treg cells isolated from myasthenia patients in comparison to those from healthy donors. Interestingly, Treg cells from prednisolone-treated myasthenia gravis patients showed an improved suppressive function compared with untreated patients, suggesting that prednisolone may play a role in the control of the peripheral regulatory network. Indeed, prednisolone treatment prevents LPS-induced maturation of monocyte-derived dendritic cells by hampering the up-regulation of costimulatory molecules and by limiting secretion of IL-12 and IL-23, and enhancing IL-10. In addition, CD4(+) T cells cultured in the presence of such tolerogenic dendritic cells are hyporesponsive and can suppress autologous CD4(+) T cell proliferation. The results shown in this study indicate that prednisolone treatment promotes an environment that favors immune regulation rather than inflammation.

Pregnancy in patients with ankylosing spondylitis: do regulatory T cells play a role?

OBJECTIVE: To investigate the numerical and functional changes of CD4+CD25(high) regulatory T (Treg) cells during pregnancy and postpartum in patients with ankylosing spondylitis (AS). METHODS: The frequency of CD4+CD25(high) T cells was determined by flow cytometry in 10 pregnant and 5 nonpregnant patients with AS as well as in 14 pregnant and 4 nonpregnant healthy controls. Pregnant individuals were investigated at the third trimester and 8 weeks postpartum. Treg cells and CD4+CD25- effector T (Teff) cells separated by fluorescence-activated cell sorting were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies, alone or in coculture, to investigate proliferation and cytokine secretion. RESULTS: The frequency of CD4+CD25(high) Treg cells was significantly higher during pregnancy than postpartum in both healthy control subjects and patients with AS. In contrast to Treg cells in healthy pregnant women, Treg cells in pregnant women with AS secreted only small amounts of interleukin-10 and showed lower suppression of tumor necrosis factor alpha and interferon-gamma secretion by CD4+CD25- Teff cells. At the postpartum time point, proinflammatory cytokine levels in the Treg/Teff cell cocultures and Teff cell monocultures were significantly higher in patients with AS than in healthy controls. CONCLUSION: Pregnancy influenced the expansion and cytokine secretion of Treg cells in both patients with AS and control subjects. However, the Treg cells of pregnant patients with AS failed to support an antiinflammatory cytokine milieu, thereby possibly contributing to the persistent disease activity of AS during pregnancy.

Enhancement of Drug-Specific Lymphocyte Proliferation Using CD25-Depleted CD3(+) Effector Cells

Background: The lymphocyte transformation test (LTT) is used for in vitro diagnosis of drug hypersensitivity reactions. While its specificity is over 90%, sensitivity is limited and depends on the type of reaction, drug and possibly time interval between the event and analysis. Removal of regulatory T cells (Treg/CD25(hi)) from in vitro stimulated cell cultures was previously reported to be a promising method to increase the sensitivity of proliferation tests. Objective: The aim of this investigation is to evaluate the effect of removal of regulatory T cells on the sensitivity of the LTT. Methods: Patients with well-documented drug hypersensitivity were recruited. Peripheral blood mononuclear cells, isolated CD3(+) and CD3(+) T cells depleted of the CD25(hi) fraction were used as effector cells in the LTT. Irrelevant drugs were also included to determine specificity. (3)H-thymidine incorporation was utilized as the detection system and results were expressed as a stimulation index (SI). Results: SIs of 7/11 LTTs were reduced after a mean time interval of 10.5 months (LTT 1 vs. LTT 2). Removal of the CD25(hi) fraction, which was FOXP3(+) and had a suppressive effect on drug-induced proliferation, resulted in an increased response to the relevant drugs. Sensitivity was increased from 25 to 82.35% with dramatically enhanced SI (2.05 to 6.02). Specificity was not affected. Conclusion: Removal of Treg/CD25(hi) cells can increase the frequency and strengths of drug-specific proliferation without affecting specificity. This approach might be useful in certain drug hypersensitivity reactions with borderline responses or long time interval since the hypersensitivity reaction. © 2014 S. Karger AG, Basel.

Medikamentenallergie: Diagnostische Möglichkeiten mit In-vitro-Methoden

Bei Medikamentenallergien kommt es zu Immunreaktionen, die gegen das Medikament gerichtet sind und klinische Symptome verursachen. Man unterscheidet zwischen Hapten- und p-i-bedingten Reaktionen, wobei letztere nur für T-Zell-Reaktionen nachgewiesen wurden. Die häufigsten Immunmechanismen, welche Medikamentenallergien zugrunde liegen, sind Vermehrung von spezifischen IgE-produzierenden B- und/oder spezifischen T-Zellen. IgG-vermittelte Reaktionen, die z.B. eine hämolytische Anämie verursachen können, sind selten. Spezifische IgE können mittels CAP-Technologie nachgewiesen werden. Das Medikament muss an eine Trägersubstanz covalent gebunden werden, was den Nachteil mit sich bringt, dass die entsprechende Bindungsstelle im Medikament für IgE nicht erkennbar ist. Der Basophilenaktivierungstest (BAT) arbeitet meist mit freiem, ungebundenem Medikament. Wie die IgE-Vernetzung stattfindet, ist allerdings unklar. Beide Teste sind nicht genügend sensitiv um den In-vivo-Test (Prick oder i.d.) zu ersetzen. Bei T-Zell-Reaktionen wird in vitro meist die Proliferation der durch das Medikament stimulierten T-Zellen erfasst. Die Blutzellen (antikoaguliertes Blut) sollte innerhalb von 24 h im Labor zur Verarbeitung ankommen, wo die Zellseparation durchgeführt wird, um die Zellen mit dem Medikament zu stimulieren. Diese Stimulation kann durch Messung von Aktivierungsmarker (mittels Flow-Zytometrie), sezernierter Zytokine (ELISA) oder 3H-Thymidin Einbau in die sich teilende Zellen (Lymphozytentransformations- Test, LTT) erfasst werden. Am meisten Erfahrung liegt für den LTT vor. Die Sensitivität wird bei eindeutigen Fällen auf 50 – 70% geschätzt, hängt aber stark vom Krankheitsbild und Medikament ab. Schwere makulopapulöse Reaktionen und DRESS (Drug Rash with Eosinophilia and Systemic Symptoms), AGEP (acute generalized exanthematous pustulosis) sind meist positiv im LTT, aber schwere bullöse Reaktionen (SJS/TEN; Stevens-Johnson Syndrom/toxische epidermale Nekrolyse) werden besser mittels Zytotoxteste und Zytokinsekretion erfasst, da eine T-Zell-Proliferation weniger prominent ist. Trotz der limitierten Sensitivität sind diese Teste gut geeignet um Kreuzreaktionen zu erfassen, bzw. für mechanistische Studien. Da Provokationsteste bei verzögerten Medikamentenallergien nicht zur Verfügung stehen (es ist unklar, wie lange und wie hoch dosiert man das Medikament bei verzögerten Reaktionen geben muss), werden diese Teste in Zukunft eher mehr eingesetzt werden. Wichtig ist, dass sie selten falsch positiv sind, und ein positives Resultat als relevant angesehen werden kann. Für die Abklärung seltener IgG-vermittelter Reaktionen kann man einen modifizierten Coombs-Test versuchen.

Biologika – Genese und Charakteristika immunvermittelter Nebenwirkungen

Die Therapie immunvermittelter und maligner Erkrankungen befindet sich im Umbruch. Zunehmend kommen gezielt mit molekularen Zellstrukturen interagierende therapeutische Proteine, sogenannte Biologika oder Biopharmaka, zum Einsatz. Biologika sind in der Regel Antikörper oder Fusionsproteine und werden gentechnologisch nach dem Vorbild körpereigener Strukturen in zellulären Expressionssystemen, wie zum Beispiel Hamster-Ovarzellen, hergestellt und in hochreiner Form verabreicht. Biologika haben mit den klassischen kleinmolekularen Medikamenten, den sogenannten Synthetika, sowohl in der Wirkungsweise wie auch hinsichtlich der Nebenwirkungen nur noch wenig gemein. Der zunehmende Einsatz von Biologika im klinischen Alltag stellt eine Herausforderung für die anwendenden Ärzte dar, die nebst den neuen Wirkmechanismen auch den Umgang mit komplexen Nebenwirkungsmustern von direkt mit dem Immunsystem interagierenden Substanzen kennen und behandeln lernen müssen. Akute Infusionreaktionen (infusion-related reaction, IRR) stellen mit einer Inzidenz von 0,1 – 3% die häufigste Nebenwirkung dar. Interessanterweise handelt es sich in den meisten Fällen nicht um IgE-vermittelte Reaktionen und lassen sogar eine Reexposition zu. Während regulatorische Behörden und die Pharmaindustrie sich vor allem auf anti-drug-antibodies (ADA)-vermittelte Nebenwirkungen konzentrieren, wollen wir hier einen Überblick über das Nebenwirkungsspektrum und mögliche Abklärungsalgorithmen geben.

Mechano-Biologie von bovinen Bandscheiben belastet unter Kompression und Torsion

Einleitung: Bandscheiben wirken als Schockabsorbierer in der Wirbelsäule und auf diese wirken meistens komplexe Kräfte, zusammengesetzt aus Kompression, Torsion und Flexion. Die biomechanishe Umgebung einer Bandscheibe ist denn auch geprägt von komplexen Belastungen. Die Forschung über die in vitro Bandscheibenbiologie hat sich bisher um die axiale Kompression konzentriert, wobei die Bedeutung von Torsion und insbesondere dem Zusammenspiel von Kompression und Torsion (="Twisting") praktisch noch nie untersucht wurde an lebenden Organkultur-Explantaten. Wir präsentieren neue mechanobiologische Daten über die Überlebenswahrscheinlichkeit von Bandscheibenzellen kultiviert in einem neuartigen, kompakten Design eines bi-axialen Bioreaktors, um die Bedeutung von Kompression und Torsion zu verstehen. Material/Methode: Bovine Schwanzbandscheiben mit den Endplatten wurden isoliert wie bereits beschrieben [2] und mechanische Belastung wurde angewendet mit einem 2 DoF Bioreaktor für 14 Tage [3]. Die Bandscheiben wurden in verschiedene Belastungsgruppen eingeteilt: 1) Keine Belastung (NL), 2) zyklische Kompression (CC) [8h: axiale Kompression mit 0.6 ± 0.2 MPa, 0.2 Hz], 3) zyklische Torsion (CT) [8h: ± 2° torsion, 0.2 Hz, 0.2 MPa compression], 4) zyklische Kompression und Torsion (CCT) [8h: 0.6 ± 0.2 MPa, 0.2 Hz & ± 2° torsion, 0.2 Hz]. Das Bandscheibengewebe wurde mit LIVE/DEAD gefärbt und miteinem konfokalen Mikroskop visualisiert um die Überlebensrate zu bestimmen. Zell Apoptosis wurde quantifiziert mit einem Caspase 3/7 Kit normalisiert zum totalen Proteingehalt (Bradford). Relative Gen-Expression von wichtigen Genen für die Bandscheibe wurde bestimmt von anabolischen, katabolischen und inflammatorischen Genen mittels real-time RT-PCR. Die Morphologie der Bandscheibenzellen wurde mittels Histologie bestimmt. Ergebnisse: Die Resultate zeigten einen starken Abfall der Zellüberlebenswahrscheinlichkeit im Zentrum der Bandscheiben, dem Nulceus Pulposus (NP), i.e. 10%, in der Gruppe mit CCT. Hingegen die Überlebenswahrscheinlichkeit im Annulus fibrosus (AF) war stabilisiert bei über 60% im NP und im AF in allen anderen Gruppen (Fig 1). Apoptotische Aktivität war statistisch signifikant erhöht in der CC-Gruppe, aber nicht in der CCT-Gruppe, was die Vermutung nahe legt, dass der erhöhte Zellverlust im NP nicht mit Apoptose sondern mit Nekrose erklärt werden kann. Die Gen Expression der anabolischen Gene COL1, COL2 und Biglycan war signifikant erhöht im AF in der CCT Gruppe, ebenfalls waren Remodeling-Gene angeschaltet wie ADAMTS4 und MMP-13 in der CCT Gruppe (Fig. 2). Der Glykosaminoglykan (GAG) Gehalt war generell im AF erhöht in den Gruppen unter mechanischer Belastung, jedoch nicht statistisch signifikant. Schlussfolgerung: Zyklische Torsion kombiniert mit zyklischer Kompression waren in dieser Studie erfolgreich und nach unserem besten Wissen zum ersten Mal an Bandscheibenexplantaten in einer 14- tägigen Organkultur angewendet worden in einem dafür speziell konzipierten Bioreaktor. Die Resultezeigten überraschend einen negativen Effekt bei physiologischen Parametern, was die Belastung (0.6MPa ± 0.2MPa) und die Torsion (± 2°) angeht. Dieser negative Effekt des "Twistings" auf die Überlebenswahrscheinlichkeit der Zellen war jedoch nur regional im NP von Bedeutung, wohingegen im AF keine Effekte zu detektieren waren.

Effect of hay dust extract and cyathostomin antigen stimulation on cytokine expression by PBMC in horses with recurrent airway obstruction

Equine recurrent airway obstruction (RAO) is an inflammatory, obstructive airway disease induced by exposure of susceptible horses to inhaled organic dust particles. The immunological process underlying RAO is still unclear. Previous studies have shown that RAO is linked to the Interleukin-4 receptor (IL-4R) gene in one Warmblood family (F1), but not in another (F2). It has also been shown that in F1, but not in F2, RAO is associated with resistance against parasites, suggesting that this association may have an immuno-genetic basis. Therefore, we hypothesized that the T helper (h)1/Th2/regulatory (Treg) cytokine profiles of RAO-associated antigen- and parasite-antigen-stimulated peripheral blood mononuclear cells (PBMC) differ between RAO-affected and healthy horses depending on their genetic background. In our study, PBMC from 17 RAO-affected and 14 healthy control horses of F1 and F2 were stimulated for 24h with antigens relevant to RAO [hay dust extract (HDE), Aspergillus fumigatus extract (AFE) and lipopolysaccharids (LPS)]; cyathostomin extract (CE) and recombinant cyathostomin antigen (RCA) or with concanavalin A (ConA). Total mRNA levels of IL-4, IL-4R, IL-13, interferon (INF)-γ and IL-10 were examined by qRT-PCR. Stimulation with either HDE or RCA resulted in significant differences in IL-4R mRNA levels between RAO-affected and control horses in F1, but not in F2. For IL-10 mRNA expression, a significant difference between RAO-affected and control horses in F1 but not in F2 was observed only following stimulation with HDE. In contrast to HDE, stimulation with CE resulted in a significant difference of IL-10 mRNA expression level between RAO-affected horses of F2 and healthy horses of F1. No significant differences were detected upon stimulation with any of the other challenge agents. These findings indicate that the immunological response, specifically IL-4R expression, in response to hay dust and cyathostomin antigens, differs between RAO-affected and healthy horses depending on their genetic background. This study shows that analysis of PBMC reveals systemic changes associated with RAO and helps to elucidate immunological pathways involved in this disease.

[Mastitis management in Swiss dairy farms with udder health problems]

The objective of this study was to describe the udder health management in Swiss dairy herds with udder health problems. One hundred dairy herds with a yield-corrected somatic cell count of 200'000 to 300'000 cells/ml during 2010 were selected. Data concerning farm structure, housing system, milking technique, milking procedures, dry-cow and mastitis management were collected during farm visits between September and December 2011. In addition, quarter milk samples were collected for bacteriological culturing from cows with a composite somatic cell count ≥ 150'000 cells/ml. The highest quarter level prevalence was 12.3 % for C. bovis. Eighty-two percent of the pipeline milking machines in tie-stalls and 88 % of the milking parlours fulfilled the criteria for the vacuum drop, and only 74 % of the pipeline milking machines met the criteria of the 10-l-water test. Eighty-five percent of the farms changed their milk liners too late. The correct order of teat preparation before cluster attachment was carried out by 37 % of the farmers only. With these results, Swiss dairy farmers and herd health veterinarians can be directed to common mistakes in mastitis management. The data will be used for future information campaigns to improve udder health in Swiss dairy farms.

Interferon-γ Induces Expression of MHC Class II on Intestinal Epithelial Cells and Protects Mice from Colitis

Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD) and involve CD4(+) T cells, which are activated by major histocompatibility complex class II (MHCII) molecules on antigen-presenting cells (APCs). However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC) affects CD4(+) T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL)-10 receptor-blocking antibodies (anti-IL10R mAb). To assess the role of interferon (IFN)-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4(+) T-helper type (Th)1 cells - but not group 3 innate lymphoid cells (ILCs) or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4(+) T cells and forkhead box P3 (FoxP3)(+) regulatory T (Treg) cells. IFN-γ produced mainly by CD4(+) T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD.