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Exercise-induced bronchoconstriction (EIB) is a prevalent condition in elite athletes caused by transient airway narrowing during or after exercise. Young athletes nowadays start early to perform high level exercise, highlighting the need to screen for EIB in a younger population. The purpose of this review is to evaluate current evidence of pre-tests with high probability to predict a positive provocation test in young and adolescent athletes, aged 12–24 years and thus indicate whether a young athlete is at risk of having EIB. Up to now, there is no validated screening test available to increase the pre-test probability of a provocation test of EIB in young and adolescent athletes. We would recommend that a clinical guideline committee might consider the development of a flow chart to screen for EIB in adolescent athletes. It could be composed of a symptom-based questionnaire focusing on wheezing during exercise, atopic state, reversibility test (to exclude EIB with asthma) and completed with markers in blood/serum. However, more research is necessary.

Adequate nasal breathing is indispensable for athletes, and nasal symptoms have been shown to interfere with their subjective feeling of comfortable breathing and quality of life. Nasal symptoms are caused by either structural abnormalities or mucosal pathology. Structural pathologies are managed differently from mucosal disease, and therefore, adequate diagnosis is of utmost importance in athletes in order to choose the correct treatment option for the individual. Literature suggests that nasal symptoms are more prevalent in athletes compared to the general population and certain sports environments might even trigger the development of symptoms. Given the high demands of respiratory function in athletes, insight into triggering factors is of high importance for disease prevention. Also, it has been suggested that athletes are more neglectful to their symptoms and hence remain undertreated, meaning that special attention should be paid to education of athletes and their caregivers. This review aims at giving an overview of nasal physiology in exercise as well as the possible types of nasal pathology. Additionally, diagnostic and treatment options are discussed and we focus on unmet needs for the management and prevention of these symptoms in athletes within the concept of precision medicine. ispartof: ALLERGY vol:76 issue:9 pages:2716-2729 ispartof: location:Denmark status: published

Taste receptors are well known for their role in the sensation of taste. Surprisingly, the expression and involvement of taste receptors in chemosensory processes outside the tongue have been recently identified in many organs including the airways. Currently, a clear understanding of the airway-specific function of these receptors and the endogenous activating/inhibitory ligands is lagging. The focus of this review is on recent physiological and clinical data describing the taste receptors in the airways and their activation by secreted bacterial compounds. Taste receptors in the airways are potentially involved in three different immune pathways (i.e., the production of nitric oxide and antimicrobial peptides secretion, modulation of ciliary beat frequency, and bronchial smooth muscle cell relaxation). Moreover, genetic polymorphisms in these receptors may alter the patients' susceptibility to certain types of respiratory infections as well as to differential outcomes in patients with chronic inflammatory airway diseases such as chronic rhinosinusitis and asthma. A better understanding of the function of taste receptors in the airways may lead to the development of a novel class of therapeutic molecules that can stimulate airway mucosal immune responses and could treat patients with chronic airway diseases. ispartof: CELLS vol:10 issue:11 ispartof: location:Switzerland status: published

Background A high burden of lower airway symptoms is found in elite swimmers. To what extent elite swimmers suffer from upper airway symptoms and how these associate with nasal inflammation is less clear. We here aimed to evaluate upper airway symptoms and nasal inflammation in elite athletes. Methodology Elite swimmers, indoor athletes and age-matched controls were recruited. Upper airway symptoms were assessed by sino-nasal outcome test (SNOT)-22 questionnaire. Visual Analogue score (VAS) for nasal symptoms as well as neurogenic and inflammatory mediators in nasal fluid were assessed at baseline, immediately and 24-hours after sport-specific training. The effect of hypochlorite on nasal epithelial cells was evaluated in vitro. Results Baseline SNOT-22 and VAS for nasal itch and impaired smell were significantly higher in swimmers compared to controls. Nasal substance P and uric acid levels were increased in elite swimmers 24-hours after swimming compared to baseline. In elite swimmers, uric acid levels 24-hours post-exercise correlated with baseline SNOT-22. As increased symptoms and inflammation were found in swimmers but not in indoor athletes, we hypothesized that hypochlorite exposure might be the underlying mechanism. In vitro, the highest dose of hypochlorite decreased nasal epithelial cell integrity and induced release of uric acid. Conclusion Upper airway symptoms are frequently reported in elite swimmers. Intensive swimming resulted in a delayed increase of epithelial injury and neurogenic inflammation.

BACKGROUND: A defective epithelial barrier is found in patients with allergic rhinitis (AR) and asthma; however, the underlying mechanisms remain poorly understood. Histone deacetylase (HDAC) activity has been identified as a crucial driver of allergic inflammation and tight junction dysfunction. OBJECTIVE: We investigated whether HDAC activity has been altered in patients with AR and in a mouse model of house dust mite (HDM)-induced allergic asthma and whether it contributed to epithelial barrier dysfunction. METHODS: Primary nasal epithelial cells of control subjects and patients with AR were cultured at the air-liquid interface to study transepithelial electrical resistance and paracellular flux of fluorescein isothiocyanate-dextran (4 kDa) together with mRNA expression and immunofluorescence staining of tight junctions. Air-liquid interface cultures were stimulated with different concentrations of JNJ-26481585, a broad-spectrum HDAC inhibitor. In vivo the effect of JNJ-26481585 on mucosal permeability and tight junction function was evaluated in a mouse model of HDM-induced allergic airway inflammation. RESULTS: General HDAC activity was greater in nasal epithelial cells of patients with AR and correlated inversely with epithelial integrity. Treatment of nasal epithelial cells with JNJ-26481585 restored epithelial integrity by promoting tight junction expression and protein reorganization. HDM-sensitized mice were treated with JNJ-26481585 to demonstrate the in vivo role of HDACs. Treated mice did not have allergic airway inflammation and had no bronchial hyperreactivity. Moreover, JNJ-26481585 treatment restored nasal mucosal function by promoting tight junction expression. CONCLUSION: Our findings identify increased HDAC activity as a potential tissue-injury mechanism responsible for dysregulated epithelial cell repair, leading to defective epithelial barriers in AR. Blocking HDAC activity is a promising novel target for therapeutic intervention in patients with airway diseases. ispartof: Journal Of Allergy And Clinical Immunology vol:144 issue:5 pages:1242-1253.e7 ispartof: location:United States status: published

The evidence of exercise-induced bronchoconstriction (EIB) without asthma (EIBwA ) occurring in athletes led to speculate about different endotypes inducing respiratory symptoms within athletes. Classical postulated mechanisms for bronchial obstruction in this population include the osmotic and the thermal hypotheses. More recently, the presence of epithelial injury and inflammation in the airways of athletes was demonstrated. In addition, neuronal activation has been suggested as a potential modulator of bronchoconstriction. Investigation of these emerging mechanisms is of major importance as EIB is a significant problem for both recreational and competitive athletes and is the most common chronic condition among Olympic athletes, with obvious implications for their competing performance, health and quality of life. Hereby, we summarize the latest achievements in this area and identify the current gaps of knowledge so that future research heads toward better defining the etiologic factors and mechanisms involved in development of EIB in elite athletes as well as essential aspects to ultimately propose preventive and therapeutic measures.

MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8+ cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was—even after major histocompatibility complex class I upregulation—insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8−CD69+ T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Significance Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune therapy and have the advantage over mesenchymal stem cells (MSCs) of large-scale manufacturing and banking potential and thus prompt availability, it is important to understand how MAPCs interact with immune cells to validate their widespread therapeutic applicability. Cytotoxic immune effector cells play a crucial role in immune homeostasis and in the pathogenesis of some autoimmune diseases. This study assessed for the first time the in vitro influence of a clinical-grade human MAPC product (MultiStem) on the cytotoxic function of CD8+ T cells (CTLs) by evaluating the immunogenicity of MAPCs and the susceptibility of MAPCs toward CTL-mediated lysis and by analyzing the mechanism of MAPC-mediated modulation of CTL functionality. These results may represent a highly relevant contribution to the current knowledge and, in combination with the results of future phase II/III trials using MultiStem, could lead to an intriguing continuation of stem cell-based research for immunotherapy.

Several studies have already identified atopy as a risk factor for the development of exercise-induced bronchoconstriction (EIB) in adult elite athletes (1-3). Skin prick test or serum specific IgE to common aero-allergens is used to determine the patient's atopy status (4). In 2009, Bonini M et al. introduced a screening tool to predict atopy in elite adult athletes, namely the Allergy Questionnaire for Athletes or short the AQUA© questionnaire (5). This article is protected by copyright. All rights reserved. ispartof: PEDIATRIC ALLERGY AND IMMUNOLOGY vol:29 issue:6 pages:648-650 ispartof: location:England status: published

Physical exercise requires proper function of the upper and lower airways in order to meet exertional ventilatory requirements. Athletes performing frequent intensive exercise experience more sino-nasal symptoms and demonstrate objective decreases in sino-nasal function when compared with the general population. Sino-nasal dysfunction is known to interfere with sport performance. Nasal epithelial injury, neutrophilic influx, and decreased mucociliary clearance have been associated with intensive training. In this review, the authors provide a comprehensive overview of the prevalence of sino-nasal disease in athletes, the possible underlying pathophysiologic mechanisms, and a summary of diagnostic and treatment options. ispartof: IMMUNOLOGY AND ALLERGY CLINICS OF NORTH AMERICA vol:38 issue:2 pages:259-+ ispartof: location:United States status: published

SummaryBackground Data from birth cohort studies suggest that increased cord blood total IgE and reduced cord blood regulatory T cells increase the risk of developing allergic sensitization and atopic dermatitis. Objective We here addressed whether serum total IgE and hen's egg-specific IgE levels at birth and at age 1 year differed between healthy and allergic children in a Belgian birth cohort (FONIA). We furthermore studied whether these parameters as well as cord blood Foxp3/CD3γ mRNA levels might predict the allergic outcome. Methods and results Children (n = 84) were clinically assessed at the ages of 6, 12, 18, and 24 months and at 6 years. Cord blood total IgE levels above 0.35 kU/L predicted early (i.e. before or at the age of 2 years) allergy development. Presence of serum IgE antibodies to hen's egg (cut-off 0.05 Ua/mL) at the age of 1 year was associated with early as well as late (i.e. between the age of 2 and 6 years) allergy development. Cord blood Foxp3/CD3γ mRNA ratios were significantly lower in early allergic children and levels below 0.32 predicted the allergic outcome. Conclusions and clinical Relevance Low cord blood Foxp3/CD3γ mRNA ratios are highly predictive for early allergy development, whereas specific IgE levels to hen's egg white above 0.05 Ua/mL at age 1 year predict allergy development in general.

In children it is often essential to recognise serious infections at an early stage to reduce possible life-threatening complications. C reactive protein (CRP) is an acute-phase protein, secreted in response to any infection or inflammation.1 Venous blood sampling can be difficult in children in ambulatory care. A point-of-care (POC) test, provided at the bedside, presents an immediate result from a droplet of blood and is especially useful in children. Previous generations of POC CRP tests have shown good correlation with standard laboratory tests in studies in primary care and emergency departments.1–3 Measuring CRP could contribute to clinical decision-making in diagnosing serious infection.4 We determined the analytical accuracy (closeness of the agreement between the measurement results and a true value) and user-friendliness of the Afinion CRP test (on the Afinion AS100 Analyzer, Alere, USA), in children and adults. To assess analytical accuracy, we performed POC CRP tests in children (aged 1 month–18 years) admitted to an inpatient paediatric unit or attending an outpatient paediatric clinic, and in adults (aged 18–65 years) attending a general practice surgery. User-friendliness was evaluated by the participating general practitioners. This study was approved by the ethical review board of the KU Leuven, under reference ML8239. ### Afinion CRP test The Afinion CRP Test Cartridge consists of a 1.5 µL glass capillary and a reagent container. The result is available within 4 min and the measuring range for CRP is 5–200 mg/L. One physician (JYV) performed all POC CRP tests in children, executing every finger stick in a similar fashion (lateral side of …

Since the discovery of IL-17 in 1995 as a T-cell cytokine, inducing IL-6 and IL-8 production by fibroblasts, and the report of a separate T-cell lineage producing IL-17(A), called Th17 cells, in 2005, the role of IL-17 has been studied in several inflammatory diseases. By inducing IL-8 production and subsequent neutrophil attraction towards the site of inflammation, IL-17A can link adaptive and innate immune responses. More specifically, its role in respiratory diseases has intensively been investigated. We here review its role in human respiratory diseases and try to unravel the question whether IL-17A only provides a link between the adaptive and innate respiratory immunity or whether this cytokine might also be locally produced by innate immune cells. We furthermore briefly discuss the possibility to reduce local IL-17A production as a treatment option for respiratory diseases. ispartof: Clinical & Developmental Immunology vol:2013 ispartof: location:Egypt status: published

Background Tight junction (TJ) defects have recently been associated with asthma and chronic rhinosinusitis. The expression, function, and regulation of nasal epithelial TJs remain unknown in patients with allergic rhinitis (AR). Objective We investigated the expression, function, and regulation of TJs in the nasal epithelium of patients with house dust mite (HDM)–induced AR and in an HDM-induced murine model of allergic airway disease. Methods Air-liquid interface cultures of primary nasal epithelial cells of control subjects and patients with HDM-induced AR were used for measuring transepithelial resistance and passage to fluorescein isothiocyanate–dextran 4 kDa (FD4). Ex vivo transtissue resistance and FD4 permeability of nasal mucosal explants were measured. TJ expression was evaluated by using real-time quantitative PCR and immunofluorescence. In addition, the effects of IL-4, IFN-γ, and fluticasone propionate (FP) on nasal epithelial cells were investigated in vitro . An HDM murine model was used to study the effects of allergic inflammation and FP treatment on transmucosal passage of FD4 in vivo . Results A decreased resistance in vitro and ex vivo was found in patients with HDM-induced AR, with increased FD4 permeability and reduced occludin and zonula occludens-1 expression. AR symptoms correlated inversely with resistance in patients with HDM-induced AR. In vitro IL-4 decreased transepithelial resistance and increased FD4 permeability, whereas IFN-γ had no effect. FP prevented IL-4–induced barrier dysfunction in vitro . In an HDM murine model FP prevented the allergen-induced increased mucosal permeability. Conclusion We found impaired nasal epithelial barrier function in patients with HDM-induced AR, with lower occludin and zonula occludens-1 expression. IL-4 disrupted epithelial integrity in vitro , and FP restored barrier function. Better understanding of nasal barrier regulation might lead to a better understanding and treatment of AR.

SummaryOne of the theories which explain, why gastroesophageal reflux disease (GORD) may provoke cough, is the occurrence of aspiration of gastric content into the airways. The aim of the study was to assess the presence of aspiration markers: pepsin and bile acids (BA) in induced sputum in gastroesophageal reflux-related (GOR-related) chronic cough (CC) patients. Forty-one CC patients and 20 healthy controls were enrolled in the study. GORD as cause of CC was diagnosed by presence of GORD-related symptoms, gastroscopy and/or improvement of cough upon treatment with proton pump inhibitors (PPI). Patients were divided into two groups based on the response to PPI treatment. In all patients and healthy controls induced sputum was obtained and differential cell counts were calculated. Levels of pepsin and BA were measured in sputum supernatants. Pepsin was detectable in 48.8% samples in CC patients and in 60% healthy controls (p = NS). In pepsin positive samples no significant difference in pepsin concentration could be found between CC patients and control subjects. Pepsin levels in pepsin positive samples were significantly decreased in patients treated with PPI compared to non-treated patients. BA were detectable in 56% samples of CC patients and in 70% healthy controls (p = NS). BA concentration in BA positive samples in CC group was not different from healthy controls. There was also no difference when comparing patients who took PPI and those who did not. Patients characterized as PPI-responders and PPI-non-responders had similar pepsin and BA concentrations. Airway cellularity was not significantly different between groups of patients with or without pepsin or BA in induced sputum. Our results demonstrated the lack of differences in gastric content aspiration between patients with probable GOR-related CC and healthy control subjects. This might imply that the reflex cough theory may be more relevant than the reflux-associated aspiration theory in the pathophysiology of GORinduced chronic cough.

Background: Immune therapies may act in inflammatory bowel diseases (IBD) by modulating regulatory T cells (Tregs). Therefore, we investigated the effect of infliximab (IFX) therapy on Forkhead box protein3 (Foxp3) T cells in blood and intestinal mucosa from Crohn's disease (CD) and ulcerative colitis (UC). Methods: Forty patients with active IBD (23 CD / 17 UC) were treated with IFX 5 mg/kg intravenously at weeks 0, 2, 6, and each 8 weeks thereafter. Blood samples were obtained before every infusion and T-lymphocyte subsets were characterized by flow cytometry. Foxp3 expression in intestinal biopsies from 43 patients with active IBD (19 CD / 24 UC) before and after IFX infusion and from 6 controls were assessed by quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. Plasma C-reactive protein (CRP), clinical response, and endoscopic healing data were collected in parallel. Results: IFX therapy resulted in a significant and sustained relative increase of CD4+CD25+Foxp3+ Treg and of CD4+CD25−Foxp3+ Treg cells in peripheral blood (both P < 0.0001 compared to baseline), particularly in responders (both P < 0.05 compared to nonresponders). The change in CRP over time inversely correlated with the increase of CD25+Foxp3+ cells (P < 0.001, r = −0.39) and durable clinical response was associated with a sustained increase of circulating Foxp3+ cells. Surprisingly, IFX therapy downregulated mucosal mRNA and protein expression of Foxp3 in UC and CD responders (both P < 0.001) but not in nonresponders. Conclusions: IFX therapy has opposite effects in Foxp3+ Treg cells in blood and gut mucosa, which suggests a redistribution of this important T-cell subset. Inflamm Bowel Dis 2010

Summary Background Staphylococcus aureus Enterotoxin B (SEB) has immunomodulatory effects in allergic airway disease. The potential contribution of SEB to the sensitization process to allergens remains obscure. Objective In order to study the effects of staphylococcal-derived toxins on the sensitization to ovalbumin (OVA) and induction of allergic airway inflammation, we have combined the nasal application of OVA with different toxins. Methods Nasal applications of OVA and saline, SEA, SEB, toxic shock syndrome toxin (TSST)-1, protein A or lipopolysaccharide (LPS) were performed on alternate days from day 0 till 12. On day 14, mice were killed for the evaluation of OVA-specific IgE, cytokine production by mediastinal lymph node (MLN) cells and bronchial hyperreactivity (BHR) to inhaled metacholine. The effect of SEB on dendritic cell (DC) migration and maturation, and on T cell proliferation was evaluated. Results Concomitant endonasal application of OVA and SEB resulted in OVA-specific IgE production, whereas this was not found with SEA, TSST-1, protein A, LPS or OVA alone. Increased DC maturation and migration to the draining lymph nodes were observed in OVA/SEB mice, as well as an increased T cell proliferation. Bronchial inflammation with an influx of eosinophils and lymphocytes was demonstrated in OVA/SEB mice, together with hyperresponsiveness and the production of IL-4, IL-5, IL-10 and IL-13 by MLN stimulated with OVA. Conclusions Our data demonstrate that SEB facilitates sensitization to OVA and consecutive bronchial inflammation with features of allergic asthma. This is likely due to augmentation of DC migration and maturation, as well as the allergen-specific T cell proliferation upon concomitant OVA and SEB application. Cite this as: W. Huvenne, I. Callebaut, M. Plantinga, J. A. J. Vanoirbeek, O. Krysko, D. M. A. Bullens, P.Gevaert, P. Van Cauwenberge, B. N. Lambrecht, J. L. Ceuppens, C. Bachert and P. W. Hellings, Clinical & Experimental Allergy, 2010 (40) 1079–1080.

To cite this article: Huvenne W, Callebaut I, Reekmans K, Hens G, Bobic S, Jorissen M, Bullens DMA, Ceuppens JL, Bachert C, Hellings PW. Staphylococcus aureus enterotoxin B augments granulocyte migration and survival via airway epithelial cell activation. Allergy 2010; 65: 1013–1020. Abstract Background: Staphylococcus aureus enterotoxin B (SEB) has recently been postulated to be involved in the pathology of granulocyte-dominated disease. Studying the immunologic interaction between SEB and airway epithelial cells in immortalized cell lines or long-term epithelial cell cultures has obvious disadvantages. Methods: We used a novel technique of freshly isolated and purified human nasal epithelial cells (HNEC) from healthy, nonallergic individuals, which were incubated for 24 h without/with SEB at different concentrations. Chemokine production was evaluated in the supernatant using Cytometric Bead Array. The chemotactic activity of the supernatant was studied in vitro using a Boyden chamber. Survival was evaluated with flow cytometry, using propidium iodide to identify dead cells. Results: Staphylococcus aureus enterotoxin B showed a dose-dependent induction of interferon-inducible protein-10, monokine induced by interferon-γ, regulated upon activation normal T cell expressed and secreted, monocyte chemoattractant protein 1 (MCP-1) and granulocyte colony stimulating factor production by epithelial cells in vitro. The supernatant of epithelial cells had chemotactic activity for granulocytes in vitro, which was enhanced in the supernatant of SEB-stimulated epithelial cells. Reduced number of propidium iodide positive granulocytes was found in the conditions where supernatant of SEB-stimulated epithelial cells was applied. Conclusion: Staphylococcus aureus enterotoxin B exerts a direct pro-inflammatory effect on HNEC, with induction of chemokine and growth factor release, resulting in the migration and prolonged survival of granulocytes in vitro.

Haptoglobin (HP) is an acute phase protein synthesized by liver cells in response to IL-6. HP has been demonstrated to modulate the immune response and to have anti-inflammatory activities. To analyze HP's effect on autoimmune inflammation, we here studied the course of EAE induced by immunization of Hp knockout (Hp(-/-)) and syngeneic WT mice with myelin oligodendrocyte glycoprotein peptide (MOG(35-55)). Hp(-/-)mice suffered from a more severe disease that was associated with increased expression of IL-17A, IL-6, and IFN-gamma mRNA in the CNS and with a denser cellular infiltrate in the spinal cord. During the recovery phase, a significantly higher number of myeloid DC, CD8+ cells, IL-17+ CD4+ and IFN-gamma+ CD4+ cells persisted in the CNS of Hp(-/-) mice. Absence of HP affected the priming and differentiation of T cells after MOG(35-55) immunization, as levels of Th2 cytokines produced in response to MOG stimulation by Hp(-/-) T cells were reduced. These results suggest that HP plays a modulatory and protective role on autoimmune inflammation of the CNS.

Dendritic cells (DC) exert important functions both in the induction of protective T cell immunity, as well as in the induction of tolerance. The latter is essential in protection against the development of allergy. Exposure to pathogen-associated molecular patterns (PAMPs), that signal through different Toll-like receptor (TLR)s, modulates production by DC of several Interleukin(IL)-12 family members: IL-12p70, IL-23, IL-27 and IL-35. The production of those cytokines in turn differentially influences both T cell differentiation and effector T cell activation. We focus in this review on myeloid and plasmacytoid DC and their role in development of allergic diseases. In particular, we focus on differences in DC activation between healthy and allergic individuals. Furthermore we discuss the activation of DC by TLR-ligands and their differential cytokine induction. Finally, we discuss preclinical studies and potentially therapeutic options using TLR-ligands for allergic diseases.

Bronchiolitis obliterans syndrome (BOS) is the leading cause of death after lung transplantation. Treatment is challenging, as the precise pathophysiology remains unclear. We hypothesize that T(H)17 lineage plays a key role in the pathophysiology of BOS by linking T-cell activation to neutrophil influx and chronic inflammation. In a cross-sectional study, bronchoalveolar lavage (BAL) samples of 132 lung transplant recipients were analyzed. Patients were divided in four groups: stable or suffering from infection (INF), acute rejection (AR) or BOS. The upstream T(H)17 skewing (TGF-beta/IL1beta/IL6/IL23), T(H)17 counteracting (IL2), T(H)17 effector cytokine (IL17) and the principal neutrophil-attracting chemokine (IL8), were quantified at the mRNA or protein level in combination with the cell profiles. The BOS group (n = 36) showed an increase in IL1beta protein (x1.5), IL6 protein (x3), transforming growth factor-beta (TGF-beta) mRNA (x3), IL17 mRNA (x20), IL23 mRNA (x10), IL8 protein (x2), IL8 mRNA (x3) and a decrease in IL2 protein (x0.8). The infection group (n = 11) demonstrated an increase in IL1beta protein (x5), IL6 protein (x20), TGF-beta mRNA (x10), IL17 mRNA (x300), IL23 mRNA (x200) and IL8 protein (x6). The acute rejection group (n = 43) only revealed an increase in IL6 protein (x6) and IL8 protein (x2) and a decrease in IL2 protein (x0.7). Lymphocytes and neutrophils were increased in all groups compared to the stable (n = 42). Our findings demonstrate the IL23/IL17 axis to be involved in the pathophysiology of BOS potentially triggering the IL8-mediated neutrophilia. IL6, IL1beta and IL23 seem to be skewing cytokines and IL2 a counteracting cytokine for T(H)17 alignment. The involvement of TGF-beta could not be confirmed, either as T(H)17 steering or as counteracting cytokine. ispartof: American Journal of Transplantation vol:8 issue:9 pages:1911-1920 ispartof: location:United States status: published

Xenothymus transplantation under the kidney capsule in athymic rodents frequently leads to multiorgan autoimmunity. Herein, we explore whether this is an intrinsic risk of xenothymus grafting or whether it depends on the transplant technique. We developed a new technique of "venous pouch" thymus grafting (heart-xenothymus) and compared this with the conventional kidney subcapsular technique (kidney-xenothymus) in a rat-into-nude-mouse model. Whereas lethal autoimmunity developed in 90% of kidney-xenothymus recipients, all heart-xenothymus grafted mice remained completely healthy. Autoimmunity in heart-xenothymus recipients was absent despite a significantly improved T-cell generation and was associated with significantly higher CD4 + CD25 + T-cell frequencies and CD4 + CD25+ cell Foxp3 mRNA levels than those observed in kidney-xenothymus recipients. In conclusion, we describe a novel vascular pouch technique of xenothymus transplantation that prevents the development of autoimmunity in nude mice. Our data further suggest that prevention of autoimmunity is related to a superior development of regulatory T-cells.

Introduction: Exercise-induced bronchoconstriction (EIB) is more common in athletes compared to the general population. The eucapnic voluntary hyperventilation test is used to detect EIB in adult athletes. It is however unclear whether this technique is also applicable to young athletes. Methods Young athletes (basketball (n = 13), football (n = 19), swimming (n = 12)) were recruited at the start of their elite sports career (12–14 years). Eight age-matched controls were also recruited. Eucapnic voluntary hyperventilation test was performed according to ATS guidelines in all subjects. A second (after 1 year, n = 32) and third (after 2 years, n = 39) measurement was performed in a subgroup of athletes and controls. Results At time of first evaluation, 3/13 basketball players, 4/19 football players, 5/11 swimmers and 1/8 controls met criteria for EIB (fall in FEV 1 ≥10% after EVH). A ventilation rate of >85% of the maximal voluntary ventilation (MVV) is recommended by current guidelines (for adults) but was only achieved by a low number of individuals (first occasion: 27%, third occasion: 45%) However, MVV in young athletes corresponds to 30 times FEV 1 , which is equivalent to 85% of MVV in adults. A threshold of 70% of MVV (21 times FEV 1 ) is feasible in the majority of young athletes. Conclusion EIB is present in a substantial number of individuals at the age of 12–14 years, especially in swimmers. This underscores the importance of screening for EIB at this age. EVH is feasible in young elite athletes, however target ventilation needs to be adjusted accordingly.

Background Anti-tumor necrosis factor (TNF) therapy has become a standard therapy for severe inflammatory bowel diseases (IBD), but its effect on B lymphocytes is largely unexplored. In this study we investigated peripheral blood B cells, B-cell subsets, and CD40 expression in patients with IBD before and during anti-TNF therapy with infliximab (IFX). Methods Blood was taken from healthy controls (n = 52) and patients with active IBD before (n = 46) and/or during anti-TNF therapy (n = 55). B-cell markers were detected by immunofluorescent staining and FACS analysis. Patients were classified as responders or nonresponders to anti-TNF therapy. Results We found a numerical deficiency of circulating CD19 B cells, a lower activation state (CD40 expression) and lower proportions of CD5 B cells and IgMIgDCD27 preswitched memory cells among B cells in active patients with IBD before IFX therapy compared with healthy controls. IFX treatment increased CD19 B-cell numbers as well as the proportions of named B-cell subsets in responders but not in nonresponders. IFX more effectively upregulated CD40 expression in responders than in nonresponders. Restoration of B cells correlated with the biological response to therapy (C-reactive protein). Trough serum levels of IFX correlated with the number of B cells during therapy. Conclusions A lower number of circulating B cells, a low CD40 expression, and a decrease in the proportion of CD5 and in the preswitched memory subset characterize active IBD. Restoration of these abnormalities correlates with the clinical response to anti-TNF therapy. The mechanism for this effect on B cells should be further explored.

Recently, spaCBA -encoded pili on the cell surface of Lactobacillus rhamnosus GG were identified to be key molecules for binding to human intestinal mucus and Caco-2 intestinal epithelial cells. Here, we investigated the role of the SpaCBA pilus of L. rhamnosus GG in the interaction with macrophages in vitro by comparing the wild type with surface mutants. Our results show that SpaCBA pili play a significant role in the capacity for adhesion to macrophages and also promote bacterial uptake by these phagocytic cells. Interestingly, our data suggest that SpaCBA pili also mediate anti-inflammatory effects by induction of interleukin-10 (IL-10) mRNA and reduction of interleukin-6 (IL-6) mRNA in a murine RAW 264.7 macrophage cell line. These pili appear to mediate these effects indirectly by promoting close contact with the macrophages, facilitating the exertion of anti-inflammatory effects by other surface molecules via yet unknown mechanisms. Blockage of complement receptor 3 (CR3), previously identified to be a receptor for streptococcal pili, significantly decreased the uptake of pilus-expressing strains in RAW 264.7 cells, while the expression of IL-10 and IL-6 mRNA by these macrophages was not affected by this blocking. On the other hand, blockage of Toll-like receptor 2 (TLR2) significantly reduced the expression of IL-6 mRNA irrespective of the presence of pili.

Background The role of bacterial enterotoxins like Staphylococcus aureus enterotoxin B (SEB) in allergic asthma remains unknown. We used a mouse model of airway allergy to study the effects of nasal or bronchial contact with SEB on bronchial allergic inflammation. Methods The features of allergic asthma were induced in ovalbumin (OVA)-sensitized mice (days 1-13) by repeated exposures to nebulized OVA (days 33-37). Nasal or bronchial application of SEB was performed on three occasions (days 33-35-37), and the effects on bronchial inflammation, IgE titres and expression levels of mRNA for T helper type 2 cytokines and other inflammatory mediators were evaluated. Results Both nasal and bronchial SEB enhanced the allergen-induced bronchial inflammation, as reflected by more eosinophilic inflammation in the airway lumen and in bronchial tissue. Aggravation of experimental asthma correlated with higher expression of mRNA for IL-5, IL-4, IFN-gamma, IL-12 p40, eotaxin-1 and TGF-beta in bronchi. In addition, nasal SEB elevated concentrations of IL-4, IL-5 and IFN-gamma in serum and bronchial SEB increased titres of OVA-specific and total IgE in serum. Conclusion Our data illustrate the potential of both nasal as well as bronchial SEB to aggravate several features of allergic asthma in a mouse model.

Summary Background T helper (Th)2 cells play an important role in the development of IgE-mediated diseases, with local overproduction of Th2 cytokines (IL-4, IL-5 and IL-13) at the site of allergic inflammation. Furthermore, IL-10 has been suggested to play a modulatory role in the induction and maintenance of allergen-specific tolerance in human atopic diseases. Aim We studied whether circulating allergen-specific Th2 cells persist outside the season of exposure in patients mono-sensitized to birch pollen and whether healthy control individuals also have allergen-specific Th2 cells. We also studied whether IL-10-producing allergen-specific T cells can be found in circulation either in healthy controls or in allergic patients. Methods Blood was drawn outside the birch-pollen season from 15 birch-pollen-allergic patients, with seasonal respiratory symptoms and with (n=12) or without (n=3) oral allergy syndrome, and from 10 matched healthy controls. Peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with recombinant Bet v 1 allergen, control antigen tetanus toxoid (TT) and anti-CD3/CD80. In part of the cultures, rIL-4 was added in order to reinforce the allergen-specific Th2 cell responses. Results In the presence of rBet v 1, T cells from allergic patients, but not from healthy controls, produced IL-4, IL-5 and IL-13. IL-5 production by patients' T cells was further enhanced by adding more IL-4. In contrast, rBet v 1 together with IL-4-induced significant IL-10 production in control subjects but not in patients. Both Th1 and Th2 cytokines were equally induced by polyclonal stimulation in allergic patients and controls, but in the presence of IL-4, polyclonally induced IL-10 production was lower in the patient group. Conclusion rBet v 1-specific Th2 cells circulate outside the season of exposure in the blood of birch-pollen-allergic subjects but not in healthy controls. Allergen-specific T cells were also demonstrated in controls but these cells produce IL-10 when stimulated with rBet v 1 in the presence of IL-4. Our data reveal a different allergen-induced cytokine profile in birch-pollen-allergic patients vs. controls, and suggest that a regulatory mechanism involving IL-4-induced allergen-specific IL-10 production might be defective in allergic subjects.

Interaction of the CD40L (CD154) molecule on activated T cells with its receptor, CD40, on macrophages and dendritic cells (DC) provides a strong signal for interleukin (IL)-12 production. As IL-12 is the most important factor in driving Th precursor (Thp) cells into T(h)elper 1 cells, CD40-CD40L interactions strongly promote Th1 differentiation. Th2 cytokines (IL-4, IL-13, IL-10) on the other hand, are known to inhibit Th1 differentiation, and to promote either directly or indirectly, Th2 differentiation. Inhibition of lipopolysaccharide (LPS)-induced IL-12 production by IL-4, IL-13 and IL-10 is supposed to be one such mechanism. However, we here report that IL-4 and IL-13 enhance p70 IL-12 production and p40 mRNA transcription by human monocytes when the latter are stimulated trough triggering of CD40. This effect on IL-12 induction is most clear in the presence of interferon (IFN)-gamma, which upregulates CD40 expression. IL-10 potently inhibits IL-12 production. The increased IL-12 production in the presence of IL-4 and IL-13 is however, not the indirect result of a reduction in IL-10 production, but is most likely owing to a direct effect of IL-4 and IL-13. We conclude that IL-4 and IL-13 enhance rather than decrease the IL-12 production by human monocytes during interaction with T cells. This effect can potentially contribute in vivo to switching of an ongoing Th2 response towards a Th1 response and the findings also support the dominant effect of CD40/CD40L interaction on Th1 development, even in the presence of Th2 cytokines.

Interleukin (IL)-10, an immunomodulatory cytokine predominantly produced by monocytes/macrophages and T cells, inhibits several functions of dendritic cells (DC), monocytes and T cells including their cytokine production, but it stimulates B cell immunoglobulin (Ig) production and cytotoxic T lymphocyte (CTL) generation. A precise knowledge of the mechanisms that control the IL-10 production is therefore highly important for understanding the immunoregulation. The IL-10 production was studied in cultures of freshly isolated human T cells. A rise in intracellular calcium as well as the common gamma-chain containing cytokine receptor triggering or CD28 triggering were found to be important signals for IL-10 induction. CD80, CD58, rIL-12 and rIFN-alpha all had efficacious and independent costimulatory activities on the IL-10 production, while PGE2 was inhibitory. Dependence on autocrine IL-2 signalling was shown by the effects of anti-IL-2 and anti-IL-2R monoclonal antibodies (MoAb), but the IL-10 production proceeded partly IL-2-independent when CD80 provided costimulation. Sensitivity to inhibition by CsA was not removed by CD80 or CD58 costimulation and/or by addition of rIL-12 or rIFN-alpha, pointing to the absolute requirement for calcineurin activity. These data reveal important differences in the regulatory pathways between IL-10 (a cytokine-inhibitory interleukin) and IL-2 (a cytokine-inducing interleukin), which can potentially be exploited therapeutically. The fact that CsA blocks the production of IL-10, which itself has important immunosuppressive properties, should be taken into account in defining immunosuppressive treatment schedules which include the use of CsA.

SUMMARYWe analysed regulatory mechanisms involved in the production of Th2 cytokines by freshly isolated human T cells. We used an in vitro culture system in which the primary signal was provided by a cross-linking anti-CD3 MoAb presented on the Fc receptors of P815 cells. Both CD80 and CD86, expressed on transfected P815 cells, were able to provide efficient costimulation for the production of IL-4, IL-5 and IL-13. IL-2 was also highly important for induction of all three Th2 cytokines. However, differences between IL-4 on the one hand and IL-5 and IL-13 on the other hand were observed when sensitivity to cyclosporin A (CsA) was studied. CsA (an inhibitor of calcineurin phosphatase activity) strongly inhibited IL-4 production, but it did either not affect or even increased IL-5 and IL-13 production. In accordance with this, CD80 and phorbol myristate acetate (PMA) (without anti-CD3 or calcium ionophore) were sufficient to induce production of IL-5 and IL-13, but not of IL-4. The subgrouping of Th2 cytokines was further confirmed at another level on the basis of differences in cell sources: IL-4 was predominantly produced by CD4+ T cells, while IL-5 and IL-13 were produced by both CD4+ and CD8+ T cells. Thus, differences in cell sources and in the requirement of the calcium/calcineurin-signalling pathway allowed us to identify two subgroups (IL-4 and IL-5/IL-13) among human Th2-type T cell cytokines.

Rationale: Airway hyperreactivity (AHR) is a key feature of bronchial asthma, and inhalation of irritants may facilitate development of nonallergic AHR. Swimmers exposed to hypochlorite (ClO(-))-containing water show a higher risk of developing AHR. We developed a mouse model in which instillation of ClO(-) before ovalbumin (OVA) induces AHR without bronchial inflammatory cells. Objectives: To investigate the mechanisms of ClO(-)-OVA-induced nonallergic AHR. Methods: The involvement of the transient receptor potential ankyrin (TRPA)1 channel was checked in vivo by the use of TRPA1(-/-) mice and in vitro by Ca(2+) imaging experiments. The role of substance P (SP) was investigated by pretreating animals with the receptor antagonist RP67580, by replacing ClO(-) with SP in vivo, and by immunofluorescent staining of large airways of exposed mice. The role of mast cells was evaluated by exposing mast cell-deficient Kit(Wh)/Kit(Wsh) mice to ClO(-)-OVA with or without mast cell reconstitution. Measurements and Main Results: ClO(-)-OVA did not induce AHR in TRPA1(-/-) mice, and ClO(-) generates a Ca(2+) influx in TRPA1-transfected cells. Pretreatment with RP67580 reduces ClO(-)-OVA-induced AHR, although no increased SP expression was shown in the airways. SP-OVA exposure resulted in the same AHR as induced by ClO(-)-OVA. Kit(Wsh)/Kit(Wsh) mice did not develop AHR in response to ClO(-)-OVA unless they were reconstituted with bone marrow-derived mast cells. Conclusions: Induction of AHR by exposure to ClO(-)-OVA depends on a neuroimmune interaction that involves TRPA1-dependent stimulation of sensory neurons and mast cell activation

Introduction Chronic non-asthmatic cough (CC) is a clinical challenge and underlying pathophysiological mechanisms remain still not completely understood. One of the most common comorbidities in CC is gastro-oesophageal reflux disease (GORD). Airway epithelium damage can contribute to airway inflammation in CC. Aims We studied airway inflammation in patients with CC compared to healthy controls. Patients with GORD were treated with proton pump inhibitors (PPI) and cough response to PPI was evaluated. Patients and methods Sputum was induced in 41 adults with CC and 20 healthy non-smokers who were age and sex matched. We compared sputum differential cell count by cytospin and cytokine and chemokine production at the mRNA and/or protein levels by real-time (RT)-PCR and cytokine bead array (CBA), between patients with CC and healthy subjects. Furthermore we studied airway inflammation in patients with different comorbidities. Results No differences in sputum differential cell counts were observed between patients with CC and healthy subjects. Sputum monocyte chemoattractant protein-1 (MCP-1) protein levels were significantly higher in patients when compared to controls. Thymic stromal lymphopoietin (TSLP) mRNA was significantly more often expressed in sputum of patients with CC than from healthy controls. Sputum transforming growth factor (TGF)-β levels did not differ between patients and controls, but were significantly lower in the PPI responders compared to the non-responders; p=0.047. There is no evidence for impaired T helper cell (Th)1/Th2/Th17 balance in CC. Patients with reflux oesophagitis (RO) have significantly more sputum eosinophils than patients without RO. Conclusions CC is a condition presenting with different disease phenotypes. High sputum MCP-1 levels are present in a large group of patients with CC and a majority of these patients with CC have increased sputum TSLP levels, most likely produced by damaged airway epithelial cells.

Placental growth factor (PlGF) and its receptor vascular endothelial growth factor receptor 1 (VEGFR1) play an important role in pathological conditions related to angiogenesis, vascular leakage, and inflammation. This study investigated their contributions to inflammation and the formation of edema in allergic asthma. The expression of PlGF and VEGFR1 was measured in induced sputum of patients with asthma (n = 11) and healthy subjects (n = 11), and in bronchial biopsies of house dust mite (HDM)-allergic patients stimulated with HDM allergens. The effects of the endonasal administration of human PlGF-2 and PlGF deficiency on inflammation and edema were evaluated in a murine model of allergic asthma. The migration of human neutrophils in response to hPlGF-2 was tested in vitro. The expression of PlGF and VEGFR1 was significantly higher in the sputum of patients with asthma, and in Der p 1-induced PlGF in biopsies from HDM-allergic patients. PlGF was increased in the bronchi of ovalbumin (OVA)-challenged mice compared with control mice (65 ± 17 pg/mg versus 18 ± 1 pg/mg, respectively; P0.01), and VEGFR1 was expressed in bronchial epithelium, endothelium (control mice), and inflammatory cells (OVA-challenged mice). The endonasal instillation of hPlGF-2 in wild-type, OVA-challenged mice led to an increase in bronchial neutrophils, lung tissue wet/dry ratio, and IL-17. PlGF-deficient mice showed lower numbers of BAL-infiltrating neutrophils, a reduced lung wet/dry ratio, and lower production of IL-17, macrophage inflammatory protein-2, and granulocyte chemotactic protein-2/LPS-induced chemokine compared with wild-type, OVA-challenged mice. hPlGF-2 induced the migration of human neutrophils in vitro in a VEGFR1-dependent way. PlGF and its receptor VEGFR1 are up-regulated in allergic asthma and play a proinflammatory role by inducing tissue edema, and increasing tissue neutrophilia and the production of IL-17.

Background T helper 17 (Th17) cells can recruit neutrophils to inflammatory sites through production of IL-17, which induces chemokine release. IL-23 is an important inducer of IL-17 and IL-22 production. Our aim was to study the role of Th17 cells in cystic fibrosis (CF) lung disease by measuring IL-17 protein and mRNA levels and IL-22 and IL-23 mRNA in sputum of clinically stable CF patients and by comparing these levels with healthy controls. Methods Sputum induction was performed in adult CF patients outside of an exacerbation and healthy control subjects. IL-17A protein levels were measured in supernatants with cytometric bead array (CBA) and RNA was isolated and quantitative RT-PCR was performed for IL-17A, IL-22 and IL-23. Results We found significantly higher levels of IL-17A protein and mRNA levels (both: p < 0.0001) and IL-23 mRNA levels (p < 0.0001) in the sputum of CF group as compared to controls. We found very low levels of IL-22 mRNA in the CF group. The levels of IL-17 and IL-23 mRNA were higher in patients chronically infected with Pseudomonas aeruginosa (P. aeruginosa) as compared to those who were not chronically infected with P. aeruginosa. The presence of Staphylococcus aureus (S. aureus) on sputum did not affect the IL-17 or IL-23 levels. There was no correlation between IL-17 or IL-23 levels and FEV1 nor sputum neutrophilia. Conclusion The elevated levels of IL-17 and IL-23 might indicate that Th17 cells are implicated in the persistent neutrophil infiltration in CF lung disease and chronic infection with P. aeruginosa.

Summary While some probiotic strains might have adjuvant effects in the therapy for inflammatory bowel diseases (IBD), these effects remain controversial and cannot be generalized. In this study, a dltD mutant of the model probiotic Lactobacillus rhamnosus GG (LGG), having a drastic modification in its lipoteichoic acid (LTA) molecules, was analysed for its effects in an experimental colitis model. Dextran sulphate sodium (DSS) was used to induce either moderate to severe or mild chronic colitis in mice. Mice received either phosphate-buffered saline (PBS), LGG wild-type or the dltD mutant via the drinking water. Macroscopic parameters, histological abnormalities, cytokine and Toll-like receptor (TLR) expression were analysed to assess disease activity. LGG wild-type did not show efficacy in the different experimental colitis set-ups. This wild-type strain even seemed to exacerbate the severity of colitic parameters in the moderate to severe colitis model compared to untreated mice. In contrast, mice treated with the dltD mutant showed an improvement of some colitic parameters compared to LGG wild-type-treated mice in both experimental models. In addition, treatment with the dltD mutant correlated with a significant down-regulation of Toll-like receptor-2 expression and of downstream proinflammatory cytokine expression in the colitic mice. These results show that molecular cell surface characteristics of probiotics are crucial when probiotics are considered for use as supporting therapy in IBD.

Autoimmune adverse events are a concern in patients treated with blocking anti-CTLA-4-mAb for solid and hematological tumors. Patient and mouse data on the contribution of a quantitative or qualitative defect of regulatory T cells (T(reg)) in this autoimmune phenomenon are conflicting. We have previously shown that a treatment course with blocking anti-CTLA-4-mAb in murine allogeneic bone marrow chimeras induces an antileukemic response in close association with systemic autoimmunity. Here, we used this model to investigate the effect of CTLA-4-blocking therapy on the kinetics of T(reg) frequency and function. As previously published, CTLA-4-blocking treatment, initiated on day 20 after bone marrow transplantation, led to overt autoimmunity by day 35. CD4(+)Foxp3(+) T(reg) frequency was determined (flowcytometry) on day 21, 23, 25 and 35: treated chimeras showed an expansion of CD4(+)Foxp3(+) T(reg) frequencies on day 25 and 35, without a prior frequency decrease. The T(reg) expansion occurred selectively in the recipient-derived CD4+ T-cell compartment. In vitro, purified CD4(+)CD25(+)FR4(high) T(reg) from 'day 35' autoimmune and control chimeras showed equal suppressive effects towards self-antigen-specific autoimmune T cells. Purified CD4(+)CD25(high)FR4(high) T(reg) from 'day 35' treated chimeras showed increased IL-10 and IFN-gamma mRNA-expression (RT-PCR) relative to control chimeras. In this model of CTLA-4-blockade-induced autoimmunity after allogeneic bone marrow transplantation, anti-CTLA-4-mAb gives rise to a progressive expansion - without a prior transient reduction - of T(reg) cells. T(reg) of autoimmune animals do not show a defect in in vitro suppressive function but show an in vivo activated cytokine profile, suggesting that the expansion occurs as a compensatory phenomenon to control autoimmunity.

Blocking of costimulatory signals for T cell activation leads to tolerance in several transplantation models, but the underlying mechanisms are incompletely understood. We analyzed the involvement of regulatory T cells (Treg) and deletion of alloreactive cells in the induction and maintenance of tolerance after costimulation blockade in a mouse model of graft-vs-host reaction. Injection of splenocytes from the C57BL/6 parent strain into a sublethally irradiated F(1) offspring (C57BL/6 x C3H) induced a GVHR characterized by severe pancytopenia. Treatment with anti-CD40L mAb and CTLA4-Ig every 3 days during 3 wk after splenocyte injection prevented disease development and induced a long-lasting state of stable mixed chimerism (>120 days). In parallel, host-specific tolerance was achieved as demonstrated by lack of host-directed alloreactivity of donor-type T cells in vitro and in vivo. Chimerism and tolerance were also obtained after CD25(+) cell-depleted splenocyte transfer, showing that CD25(+) natural Treg are not essential for tolerance induction. We further show that costimulation blockade results in enhanced Treg cell activity at early time points (days 6-30) after splenocyte transfer. This was demonstrated by the presence of a high percentage of Foxp3(+) cells among donor CD4(+) cells in the spleen of treated animals, and our finding that isolated donor-type T cells at an early time point (day 30) after splenocyte transfer displayed suppressive capacity in vitro. At later time points (>30 days after splenocyte transfer), clonal deletion of host-reactive T cells was found to be a major mechanism responsible for tolerance. ispartof: Journal of Immunology vol:181 issue:2 pages:1034-1042 ispartof: location:United States status: published

Background Recombinant hypoallergens that display reduced allergenicity but retain T-cell reactivity represent promising candidates to improve the safety and efficacy of allergen-specific vaccines or immunotherapy. Objective The current study reports the immunologic characterization of a hypoallergenic variant of the major mite allergen Der p 1. Methods The recombinant proform of Der p 1 (ProDer p 1) was expressed in Escherichia coli (ProDer p 1 coli ), purified and characterized at the level of its secondary structure, and IgE and T-cell reactivities. Moreover, the prophylactic potential of ProDer p 1 coli vaccinations was evaluated in a murine Der p 1 sensitization model. Results After purification and refolding, ProDer p 1 coli remained aggregated with a higher β-sheet content and altered Der p 1 conformational epitopes compared with the correctly folded monomeric ProDer p 1 produced in Chinese hamster ovary cells. Both ProDer p 1 forms were able to retain the Der p 1–specific T-cell reactivity but direct ELISA, competitive inhibition, and rat basophil leukemia assays clearly showed that ProDer p 1 coli displays a very weak IgE reactivity. Mice vaccinations with aggregated ProDer p 1 adjuvanted with alum induced a T H 1-biased immune response that prevented the subsequent allergic response after Der p 1 sensitization and airway challenge with aerosolized mite extracts. Furthermore, ProDer p 1 coli treatment inhibited the development of airway eosinophilia and airway hyperresponsiveness to inhaled methacholine. Conclusion Aggregated forms of Der p 1 could represent hypoallergens suitable for the prevention of mite allergy.

Allergen inhalation by sensitized asthmatics induces an IgE and mast cell dependent bronchoconstriction and a Th2-dependent inflammatory airway reaction, mucus hypersecretion and airway hyperreactivity. The link between T cells and bronchoconstriction remains controversial. Here we analyzed allergen-induced changes in airway tone in ovalbumin-sensitized mice with established allergic airway inflammation. Inhalation of nebulized ovalbumin elicited a dose-dependent and allergen-specific increase in airway resistance and bronchial tone with a concomitant increase of lymphocytes and eosinophils in bronchoalveolar lavage fluid. A Th2 pattern of cytokine expression and increased mRNA expression of MCP-1, RANTES and VCAM-1 were demonstrated. Anti-CD4 monoclonal antibody treatment prior to provocation decreased IL-13 and VCAM-1 mRNA expression and abolished the increase in bronchial tone and the inflammatory response. We conclude that allergen inhalation in sensitized mice induces airway narrowing similar to the late asthmatic reactions in humans and that this phenomenon is based on activation of CD4+ T cells.

Xenoantibody production directed at a wide variety of T lymphocyte-dependent and T lymphocyte-independent xenoantigens remains the major immunologic obstacle for successful xenotransplantation. The B lymphocyte subpopulations and their helper factors, involved in T-cell-independent xenoantibody production are only partially understood, and their identification will contribute to the clinical applicability of xenotransplantation. Here we show, using models involving T-cell-deficient athymic recipient mice, that rapidly induced, T-cell-independent xenoantibody production is mediated by marginal zone B lymphocytes and requires help from natural killer (NK) cells. This collaboration neither required NK-cell-mediated IFN-gamma production, nor NK-cell-mediated cytolytic killing of xenogeneic target cells. The T-cell-independent IgM xenoantibody response could be partially suppressed by CD40L blockade. ispartof: BLOOD vol:110 issue:12 pages:3926-3935 ispartof: location:United States status: published

BACKGROUND: T-helper type 2 (Th2) cells play an important role in the pathogenesis of allergic diseases. Recent studies have demonstrated that allergen-specific T cells can also be found in the blood of healthy individuals. Both IL-10 and IFN-gamma might modulate the induction and maintenance of allergen-specific tolerance. AIM: To study the phenotype and functional characteristics of allergen-specific T cells in healthy non-atopic children. METHODS: Peripheral blood mononuclear cells (PBMC) from 13 symptomatic house dust mite (HDM)-allergic children and from nine matched healthy control children were stimulated with recombinant (r)Der p 2, a major allergen from HDMs. RESULTS: Stimulation with rDer p 2 resulted in Th2 cytokine production in cultures of PBMC from allergic but not from healthy children. In contrast, IL-10 and IFN-gamma were induced in PBMC cultures from both healthy and HDM-allergic children. Intracellular staining revealed that IL-10 and IFN-gamma are largely produced by the same T cells. Stimulation of T cells from healthy children with rDer p 2 also induced expression of inducible costimulator (ICOS) on a small T cell subset. CONCLUSION: Allergen-specific memory T cells from healthy non-atopic children produce IL-10 and IFN-gamma (but not Th2 cytokines) and express ICOS upon stimulation. These cells might be responsible for a normal immune balance after allergen encounter in non-atopics.

Background The role of Th2 cells (producing interleukin (IL-)4, IL-5 and IL-13) in allergic asthma is well-defined. A distinct proinflammatory T cell lineage has recently been identified, called Th17 cells, producing IL-17A, a cytokine that induces CXCL8 (IL-8) and recruits neutrophils. Neutrophilic infiltration in the airways is prominent in severe asthma exacerbations and may contribute to airway gland hypersecretion, bronchial hyper-reactivity and airway wall remodelling in asthma. Aim to study the production of IL-17 in asthmatic airways at the mRNA level, and to correlate this with IL-8 mRNA, neutrophilic inflammation and asthma severity. Methods We obtained airway cells by sputum induction from healthy individuals (n = 15) and from asthmatic patients (n = 39). Neutrophils were counted on cytospins and IL-17A and IL-8 mRNA expression was quantified by real-time RT-PCR (n = 11 controls and 33 asthmatics). Results Sputum IL-17A and IL-8 mRNA levels are significantly elevated in asthma patients compared to healthy controls. IL-17 mRNA levels are significantly correlated with CD3γ mRNA levels in asthmatic patients and mRNA levels of IL-17A and IL-8 correlated with each other and with sputum neutrophil counts. High sputum IL-8 and IL-17A mRNA levels were also found in moderate-to-severe (persistent) asthmatics on inhaled steroid treatment. Conclusion The data suggest that Th17 cell infiltration in asthmatic airways links T cell activity with neutrophilic inflammation in asthma.

CD25+CD4+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance against self and non-self. The modulatory effects of cytokines, such as interleukin 4 (IL-4) on the function of Tregs have not been explored in detail. We here report that IL-4 prevents spontaneous apoptosis and the decline of foxp3 mRNA which were found to occur during culture of isolated Tregs. Tregs exposed to IL-4 were more potent in suppressing the proliferation of naive CD4+ T cells and they better inhibited IFN-gamma production by CD4+ T cells as compared to Tregs cultured in medium. IL-4 also enhanced membrane IL-2Ralpha (CD25) expression on Tregs above the levels observed on freshly isolated cells. IL-4-mediated effects on Treg function persisted in Tregs from Stat6-/- mice, pointing to a Stat6-independent intracellular transduction pathway. In conclusion, our data suggest that the anti-inflammatory function of IL-4 could partly be mediated by effects on Tregs function.

Although immunoregulation by several types of regulatory T cells is now clearly established in mice, the demonstration of such regulatory T cells in humans has been proven more difficult. In this study we demonstrate the induction of anergic regulatory T cells during an MLR performed in the presence of blocking mAb to the costimulatory molecules CD40, CD80, and CD86. Despite this costimulation blockade, which totally blocks T cell proliferation and cytokine production, a nonproliferating T cell subpopulation was activated to express inducible costimulator (ICOS). These ICOS+ cells were anergic when restimulated with unmanipulated allogeneic stimulator cells at the level of proliferation and Th1 and Th2 cytokine production, but they did produce IL-10. These ICOS-expressing cells also blocked the capacity of reciprocal ICOS-negative cells to proliferate and to produce cytokines. ICOS+ anergic cells could suppress allogenic responses of either primed or naive T cells through inhibition of IL-2 gene transcription. Suppression was not mediated by IL-10 and did not require ICOS-ICOS ligand interaction, but depended on cell-cell contact. Thus, a subtype of regulatory T cells in human blood can be activated in the absence of costimulatory signals from CD40, CD80, and CD86, and they can be identified by expression of ICOS after activation.

4-1BB ligand (L) expressed on antigen presenting cells (APC) interacts with 4-1BB, expressed on activated T cells and this interaction costimulates T cells to secrete cytokines and to proliferate. We investigated whether 4-1BB/4-1BBL interactions might be involved in the pathogenesis of Crohn's disease (CD). In immunohistochemistry, we found 4-1BB expression on lamina propria (LP) cells in inflamed and to a lesser extend in non-inflamed gut tissue from CD patients. mRNA levels for 4-1BB were also elevated in intestinal CD tissue. In contrast, only few 4-1BB-expressing cells were found in inflamed tissue from ulcerative colitis (UC) patients and almost no positive cells were found in control intestinal tissue. 4-1BB expression was better sustained on in vitro activated lamina propria T cells from CD patients compared to controls. Finally, agonistic anti-4-1BB antibody enhanced interferon-gamma (IFN-γ) production and proliferation of lamina propria T cells from CD patients. Taken together, our data suggest that 4-1BB/4-1BBL interactions contribute to the persistence of gut inflammation in CD.

Exercise induced bronchoconstriction (EIB) is more prevalent in elite athletes compared to controls. It is however unclear how many young athletes suffer from EIB.

CD58 is the ligand for the CD2 molecule on human T cells and has been shown to provide a co-stimulatory signal for T cell activation. However, its physiological role is still unclear. We studied the effects of co-stimulation by CD58 on the production of T(h)1-type (IL-2- and IFN-gamma) or T(h)2 type (IL-4, IL-5 and IL-10) cytokines in an in vitro culture system of purified human T cells with CD58-transfected P815 cells and with anti-CD3 as the primary stimulus. Co-stimulation of T cells by CD58 potently induced IL-10 and IFN-gamma production (at the protein and at the mRNA level), and transforming growth factor-ss production (at the mRNA level), comparable to what can be found in CD80 co-stimulated T cell cultures. In contrast, we found low to absent IL-2, IL-4, IL-5, IL-13 and tumor necrosis factor-alpha production after CD58 co-stimulation, and this was not due to suppressive effects of endogenously produced IL-10. CD80 co-stimulation strongly induced all these cytokines. Intracellular staining for cytokine expression revealed the existence of a T cell subpopulation induced by CD58 co-stimulation to produce both IFN-gamma and IL-10. We furthermore found that the selective cytokine profile induced by CD58 co-stimulation is further accentuated by rIL-12 and by rIFN-alpha. Using cyclosporin A as an inhibitor of the calcineurin enzyme, we could show that production of all cytokines in this system is calcium dependent. CD58 co-stimulation thus induces a cytokine pattern corresponding to that described for T regulatory (T(r)) 1 cells and to the pattern reported to be induced by the newly identified B7 family member, B7-H1.

SUMMARYIL-4 is a pleiotropic cytokine which is involved in the development of atopic diseases. Only limited data exist on IL-4 production in humans, and the relative contribution to atopy of either unbalanced IL-4 production, or increased IL-4-responsiveness of target cells, is still unknown. The use of a MoAb to the IL-4 receptor α-chain (IL-4Rα) enabled us to demonstrate that IL-4 production in vitro is usually underestimated, due to in vitro consumption, even in cultures of purified T cells. When IL-4 consumption was blocked, it became evident that CD80 and CD86 both provide effective costimulatory signals for high IL-4 production. Moreover, we found that even stimulation with a soluble antigen (tetanus toxoid) induces IL-4 production by T cells from healthy non-atopic donors. Both sets of data imply that IL-4 is not required for IL-4 production by memory and/or effector T cells. Our data further show that endogenous IL-4 activity modulates IL-10 and interferon-gamma production by T cells in opposite directions. The use of this receptor-blocking antibody will thus be helpful for in vitro studies on IL-4 regulation. Consumption of IL-4 by different cell types during in vitro cultures might have interfered with previous attempts to quantify IL-4 production by human T cells.