IMMUNOLOGY

The Influence of Trappin-2 on the Phagocytic Capacity of Macrophages

A.F.Lipka

Leiden University

Introduction: Trappin-2 is an inhibitor of human neutrophil elastase with antimicrobial properties present in the lung. Previous research has shown a protective effect of trappin-2 in animal models of pulmonary infection, due to various mechanisms. This study aimed to investigate whether trappin-2 could also enhance phagocytosis of Pseudomonas aeruginosa by macrophages.

Material and Methods: The effect of trappin-2 on clearance of P. Aeruginosa was assessed by in vitro macrophage antibiotic assays using murine bone-marrow derived macrophages, MH-S murine alveolar macrophages and human alveolar macrophages by co-incubation with 100 nM trappin-2 and P. Aeruginosa.

Results: Macrophage antibiotic assays using murine bone-marrow derived macrophages and human alveolar macrophages did not result in significant differences between treatments. In experiments using murine alveolar macrophages, pre-incubating P. Aeruginosa with trappin-2 resulted in a non-significant but consistent trend towards a synergistic effect of both trappin-2 and murine alveolar macrophages on bacterial clearance. Incubating bacteria with trappin-2 and macrophages resulted in a decrease of bacteria of 43.6% compared to macrophages alone whereas adding trappin-2 to bacteria without macrophages showed a 23.1% reduction in bacteria (p>0.05).

Conclusion: If reproducible in repeat experiments, these data support the concept that trappin-2 could act as an opsonin and therefore be a target for new therapeutic strategies in pulmonary infection.

Marked Differences in Fine Specificity and Isotype Usage of the Anti-Citrullinated Protein Antibody in Health and Disease

A. Ioan -Facsinay², A. Willemze², D.B. Robinson³, C.A. Peschken³, J. Markland⁴, D. van der Woude², B. Elias³, H.A. Menard⁵, M. Newkirk⁵, M.J. Fritzler¹, R.E.M. Toes², T.W.J. Huizinga², H.S. El-Gabalawy³

¹University of Calgary
²LUMC
³University of Manitoba
⁴University of Saskatchewan
⁵McGill University Health Center

Introduction: Anti–citrullinated protein antibodies (ACPAs) display high association with rheumatoid arthritis (RA) and are implicated in its pathogenesis. The presence of ACPAs is known to precede the onset of RA. In order to identify the features that could confer its pathogenicity, we extensively characterized this antibody response in a unique North American native population of patients with RA and their unaffected relatives.

Material and Methods: The levels of IgA, IgM, and IgG ACPAs, as well as IgM and IgA rheumatoid factor (RF), were measured in serum samples obtained from 81 patients with RA and 195 of their unaffected relatives. The isotype distribution, the fine specificity of the ACPA response, and its association with RF were compared in health and disease.

Results: ACPA positivity was observed in 19% of the healthy relatives and 91% of the patients with RA. ACPA isotype usage was strikingly lower in unaffected relatives than in patients with RA (1–2 versus 5–6 isotypes). Fine specificity studies showed that reactivity to citrullinated fibrinogen and vimentin was present in sera from patients with RA, while it was virtually absent in their unaffected relatives. Finally, the ACPA and RF responses were associated in patients with RA but were discordant in their healthy relatives. Extended analyses revealed that the presence of ACPAs was associated with RA irrespective of RF status, while the association of RF with disease relied on its interaction with ACPAs.

Conclusion: The fine specificity and isotype usage of the ACPA response are qualitatively different in health and disease. Epitope spreading and expansion of the isotype repertoire might be necessary for development of RA, and this could be facilitatedby the presence of RF antibodies

The Artificial Thymus

H. Kawamoto¹, W. van Ewijk², N. Grotenhuis²

¹RIKEN research center for allergy and immunology
²LUMC

Introduction: The thymus is an organ that produces T cells for immunity. However, sometimes the thymus does not work properly, or is completely absent. In case of autoimmune diseases or immunodeficiency, treatment could consist of implanting an artificial thymus, that produces new T cells, makes new immunity, or stops the auto-immune reactivity. An artificial thymus could also be a possible treatment for cancer, with the purpose of producing new T cells that can attack cancer. Therefore, the aim of the present research was to construct an artificial thymus.

Material and Methods: For the production of an artificial thymus, stromal cells (for example thymic epithelial cells) are required, and therefore a mesenchymal cell-line was used, TST-4/DLL1.
It has been shown before that in the development of an artificial lymph node in vivo, a collagen sponge works as an efficient scaffold. In the present study we tested whether a collagen sponge was also suitable for the formation of an artificial thymus in vivo. Also we tested a re-aggregate artificial thymus. All artificial thymuses were transplanted in mice.

Results: We found that the collagen sponge can support T cell development, but not very efficiently. A re-aggregate lobe however, is more efficient to construct an artificial thymus. We found that a re-aggregate lobe of TST-4/DLL1 could restore T cell differentiation in nude mice, lacking the thymus. Transplantation of this re-aggregate lobe under the kidney capsule of this nude mice resulted after 14 days in the presence of mature T cells.

Conclusion: It is possible to construct an artificial thymus, however more research is needed to prolonging the effectiveness of the artificial thymus. We hope we have taken some first steps for a new treatment for auto-immune diseases and maybe for cancer and HIV.

Analysis of High Resolution Computed Tomography Findings in Patients Suffering from Lung Sarcoidosis

A. Ragaji, M. Strunjas, S. Pena

Medical Faculty University Novi Sad

Introduction: Sarcoidosis is a multisystemic, noncaseating granulomatous disease of unknown origin. Most commonly involved are the lung parenchyma, pleura, and hilar lymph nodes.
Aim: The aim of this research was the analysis of high resolution computed tomography (HRCT) results from patients suffering from lung sarcoidosis.

Material and Methods: Disease histories, CT and HRCT from the electronic archives of patients suffering from lung sarcoidosis were processed in a retrospective study. The study involved 52 patients (aged 45,3 +/- 10,9 years), 34 (65,38%) female gender (aged 47,4 +/- 10,8 years) and 18 (34,62%) male gender (aged 41,4 +/- 10,5 years). During the course of this study the analysis was made concerning presence and frequentness CT and HRCT signes characteristic for lung sarcoidosis, also concerning their distribution in the craniocaudal (CC) and centro-peripheral (CP) direction, and concerning the symmetry of the lesions.

Results: The characteristical CT findings of bilateral hilar and paratracheal swelling of lymph nods, without the presence of changes in the lung parenchyma was found in 33 (63,46%) patients. Typical diffuse reticulonodular lesions in the interstitium of the lung were found in 43 (82,69%) patients. Definitive lesions (individual cysts, multiple cysts, tractional bronchiectasias, conglomerate masses of fibrous tissue and dislocation of the bronchus), cosequences of advanced fibrosis, were found in 15 (28,85%) patients. Craniocaudal predominance ( registered with 19 (36,54%) of patients ) and symmetry of lesions ( found in 35 (67,31%) examined cases) are a common finding in patients suffering from lung sacoidosis. The centro-periferal predominance on the distribution of lesions is an extremely rear radiological picture in lung sarcoidosis patients (found in 2 (3,85%) patients), as are excavated lesions and pleural effusions (not found in any of the examined cases).

Conclusion: High resolution computed tomography has a great siginficance in diagnosing sarcoidosis because it is a more sensitive and more specific method then standard radiography of the chest. The primary role of HRCT is to differentiate between patients with typical sarcoidosis lesions and those with other diffuse interstitial lesions.

The Role of IL-10 Producing Regulatory B Cells in Protection for Allergic Disease during Chronic Schistosomiasis

L.E.P.M. van der Vlugt, H.H. Smits

LUMC

Introduction: Allergic disorders, characterized by allergen-specific T helper type (Th) 2 cell immune responses, are a major health concern in the Western world. Epidemiological studies demonstrated that chronic Th2-skewed helminth infections are negatively associated with allergic disorders and may strongly suppress T cell responses to bystander antigens, such as allergens. The cytokine IL-10 is central to helminth-induced immune regulation of allergic responses and is in part secreted by helminth-induced regulatory T (Treg) cells but may also be produced by a subset of B cells. IL-10 producing B cells have a great potential in regulating T cell mediated inflammatory responses and therefore are named regulatory B cells (Bregs). The role of Bregs has been mainly studied in murine models of hyperinflammatory Th1 responses such as rheumatoid arthritis and experimental autoimmune encephalomyelitis. Several B cell subsets could be candidates to develop and act as regulatory B cell, namely CD5+ B1, follicular (FO), marginal zone (MZ), and transitional B cells.

Material and Methods: We sought to address the question whether IL-10 producing B cells play a role in the helminth-induced immunoregulatory processes during the chronic stage of schistosome infections. Furthermore, we investigated which B cell subset has the capacity to develop into Breg cells. We infected mice with helminth Schistosoma mansoni and sensitized and challenged the mice with ovalbumin (OVA) to induce allergic airway inflammation. To this end, mice were analyzed at 8 or 16 weeks, representing the acute or chronic phase of infection. We purified B cells from the spleen and sorted them into FO and MZ B cells. Next, we investigated the IL-10 production of different B cell subsets, their capacity to induce FoxP3+ Treg cells, and the expression of certain surface markers.

Results: Using IL-10-/- B cell deficient mice, we demonstrated that the helminth-mediated protection against OVA-induced allergic airway inflammation was crucially dependent on the presence of IL-10 producing B cells. Most notably, we detected that MZ B cells were the highest IL-10 producers during chronic schistosomiasis, in particular in response to TLR ligands PGN, Poly I:C, CpG and LPS plus helminth antigens. In addition, MZ B cells also induced the highest percentage of FoxP3+ T cells in vitro and in vivo. Furthermore, we observed that CD5+ B1, FO and MZ B cells were different in their expression of certain surface markers.

Conclusion: Taken together, these findings underline an important role for schistosome-induced regulatory MZ B cells in the protection against allergic airway inflammation.

Allergy to Animal Fur and Feathers in Patients with Different Allergic Diseases

N. Ukleja, L. Sokolowski, E. Gawronska-Ukleja, Z. Bartuzi

Bydgoszcz

Introduction: Establishing the frequency of allergy to animal fur and feathers in patients with bronchial asthma, seasonal rhinitis and other allergic diseases.

Material and Methods: The research was carried out in 160 patients (105F and 55M) aged 12-64 treated in the Outpatients Clinic of Allergic Diseases. Every patient was interviewed for allergy. Every patient had:

1. Spirometry at rest and bronchodilatatory test
2. Skin-Prick test for inhaled allergens and animal fur and feathers.

Results: In 160 patients we found positive Skin Prick tests for dogs dander in 41 cases (25,6%), cats in 40 cases (25%), hamster in 15 cases (9,3%), horse in 10 cases (6,25%), guinea pig in 8 cases (5%) and feathers in 15 cases (9,3%). Positive result for at least one allergen in 69 cases (43,13%). In the group of patients 25 suffered from seasonal allergic rhinitis, 56 suffered from bronchial asthma, 32 suffered both from asthma and seasonal allergic rhinitis and 47 suffered from other allergy. In 25 patients with seasonal allergic diseases positive Skin Prick test for dogs dander in 5 cases (20%), cats in 5 cases (20%), hamster in 2 cases (8%), horse in 0 cases (0%), guinea pig in 1 cases (4%) and feathers in 3 cases (12%). In 56 patients with bronchial asthma positive Skin Prick test for dogs dander in 11 cases (19,6%), cats in 9 cases (16,07%), hamster in 5 cases (8,9%), horse in 5 cases (8,9%), guinea pig in 6 cases (10,7%) and feathers in 5 cases (8,9%). In 32 patients with bronchial asthma and seasonal allergic rhinitis positive Skin Prick test for dogs dander in 16 cases (50%), cats in 15 cases (46,9%), hamster in 2 cases (6,25%), horse in 2 cases (6,25%), guinea pig in 0 cases (0%) and feathers in 1 cases (3,125%). In 47 patients with other allergies frequency of allergy to dogs dander in 9 cases (19,15%), cats in 11 cases (23,4%), hamster in 6 cases (12,77%), horse in 3 cases (6,38%), guinea pig in 1 cases (2,13%) and feathers in 6 cases (12,77%).

Conclusion:

1. In the group of patients with bronchial asthma and seasonal allergic rhinitis allergy to dog and cat dander is more frequent then in other groups (50% and 46,9%)
2. Allergy to animals is a huge problem among patients with allergic diseases (43% with positive Skin Prick test for at least one animal allergen).

FoxP3+ Regulatory T Cells in Childhood Allergic Rhinitis and Asthma

S. Arshi, N. Javahertarash, R. Zarrinfard, N. Akbar Pur, F. Jalali, F. Mohammad Beygi

Iran University of Medical Science, Tehran, Iran

Introduction: Regulatory T cells (Tregs) play a central role in the pathogenesis of immune mediated disease. We investigated CD4+FoxP3+ and CD4+CD25high regulatory T cells in children with allergic rhinitis and asthma, and compared their prevalence to that in healthy children. We also aimed to describe the relationship between the number of Tregs and disease severity. The prevalence of activated lymphocytes, the target cells of Tregs was also tested.

Material and Methods: 8 children with allergic rhinitis, 8 with mild asthmatic symptoms and 6 with moderate-severe asthma were enrolled along with 13 healthy age-matched children (median age, range: 9.5 [5-18] years). Children with moderate-severe asthma were also reassessed one month later when their symptoms improved. Peripheral blood samples were taken, and mononuclear cells were isolated. Lymphocytes were then detected with flow cytometry using specific antibodies.

Results: The prevalence of CD4+FoxP3+ cells in patients with allergic rhinitis and mild asthma was comparable to that in healthy controls (median, quartile: 2.54 [1.63-3.56]%; 1.90 [1.07-2.36]%; 1.68 [0.88-3.24]%, respectively), but it was significantly increased in patients with moderate-severe asthma (median, quartile: 4.18 [3.35-6.27]%, p = 0.027). Furthermore, our trend analysis revealed an association between disease severity and prevalence of CD4+FoxP3+ cells (p = 0.01). In patients with moderate-severe asthma after one month of therapy, FoxP3 values showed a tendency to decrease. The prevalence of activated (CD25+, CD62L+ or HLA-DR+) CD4+ and CD8+ lymphocytes was comparable in patient and control groups. No association between activated lymphocytes and FoxP3 values was revealed.

Conclusion: In this study we found that the prevalence of CD4+FoxP3+ regulatory T cells is increased in children with moderate-severe asthma. The shift of Th1/Th2 ratio to the Th2 direction is a well known phenomenon in allergy. The explanation for this skewness has been unclear. Increased Treg cell function may be a contributing factor, as the inhibitory effect of this cell type is more pronounced on Th1 than on Th2 type CD4+ cells. We could not detect an association between CD4+FoxP3+ and CD4+CD25high cells, which supports the notion that CD25 is rather an activation marker and not a specific marker of Tregs, and no clear distinction can be made between CD25+ and CD25high cells. Hence, the prevalence of CD4+CD25high cells does not necessarily reflect that of Tregs. We also found a positive correlation between increasing Treg prevalence and severity of disease. Therefore our results support the contribution of Tregs to severe asthma in childhood.

Aeroallergens in Allergic Rhinitis Patients, in Tehran, Iran (Comparing Adult and Children)

M. Förster¹, Dr. C. Sina¹, Dr. O. Gavrilovav¹, S. Derer¹, F. Hildebrandt¹, B. Raabe², Prof. Dr. Rosenstiel¹

¹Institute for Clinical Molecular Biology
²Institute of Biochemistry

Introduction: Inflammatory bowel diseases (IBD) — Crohn disease (CD) and ulcerative colitis (UC) — are disorders of unknown etiology characterized by chronic relapsing-remitting inflammation of the gastrointestinal tract. Pathophysiological and genetic evidence points to an important role of intestinal barrier function in the initiation and perpetuation of the disease. Molecular danger signals attract neutrophilic granulocytes (PMNs) to sites of infection. The G protein coupled receptor (GPR) 43 recognizes short chain fatty acids (SCFA), i.e. propionate and butyrate and is abundantly expressed on PMNs. The functional role of GPR43 activation for the orchestration of immune responses in vivo is unclear.

Material and Methods: We examined dextrane sodium sulphate (DSS)-induced acute and chronic intestinal inflammatory responses in wild-type and Gpr43-deficient mice. Clinical signs in a disease activity index, histological scoring and cytokine production in colonic tissue via ELISA-technique assessed the severity of inflammation in the colon. Chemotaxis of PMNs isolated from wildtype and Gpr43-/- mice was assessed using a transwell cell chemotactic assay (Boyden chamber).

Results: In vivo a reduced invasion of PMNs is seen in acute colitis, which is paralleled by increased mortality due to septic complications. In chronic DSS colitis, Gpr43-/- animals also show a diminished PMN migration into the intestinal mucosa but are protected from inflammatory tissue destruction. No difference in PMN migration and cytokine secretion could be detected in a sterile inflammatory model (air pouch). Ex vivo experiments show that GPR43-induced migration is dependent on the activation of the protein kinase p38α and that this signal acts in co-operation with the chemotactic cytokine KC.

Conclusion: The results indicate a critical role for GPR43-mediated recruitment of PMNs for the containment of bacterial translocation in the intestine, but also emphasize the bipotential role of PMNs mediating tissue destruction in chronic intestinal inflammation.

Aberrant Splicing of Artemis in a SCID Patient can be Restored in vitro

H. IJspeert¹, A.C. Lankester², C. Weemaes³, A. Warris³, W. Wiegant², M.C. van Zelm¹, M.J.D. van Tol², D.C. van Gent¹, J.J.M. van Dongen¹, M. van der Burg¹

¹Erasmus MC, Rotterdam
²LUMC, Leiden
³UMC St. Radboud, Nijmegen
The Netherlands

Introduction: T-ALL is a T-cell malignancy affecting children and adolescents, caused by cooperation of mutations affecting proliferation, survival, cell cycle, and differentiation.

To detect novel genomic rearrangements, we performed 1 Mb resolution array CGH with extra probes covering candidate oncogenes, as the RTKs (Receptor Tyrosine Kinase). A new fusion gene involving an RTK which functions by kinase activity and nuclear translocation, was detected in one cell line. The other gene involved is a transcription factor expressed in lymphocytes that regulates development and differentiation. Specific screening for this fusion transcript showed it’s presence in 31% in a higher number of T-ALL cell lines.

Materials and Methods: constructions: we generated the sequence of the different fusion variants found in the cell lines by PCR followed by clonation into the MSCV-puromycin vector. Transfection of Hek293T cells was done with Genejuice reagent and the expression and activity level of the RTK was analyzed by Western Blotting. Screening in patients: Samples from 25 patients with T-ALL were screened for the presence of the fusion transcript at RNA level. Cell culture: T-ALL cell lines were cultured in RPMI-1640 supplemented with 20% FCS. We cultured BaF3 cells in RPMI-1640 medium supplemented with 10 % FBS and 1 ng/ml mouse IL3.By retroviral transduction and subsequent puromycin selection we made BaF3 cells express the different constructs. To test IL3-independency, stable BaF3 cell lines were washed three times in PBS and cultured in IL3-free medium. The number of viable cells was counted with a Vi-cell XR cell viability analyzer (Beckman Coulter, Fullerton, CA).
Results: The new fusion transcript was detected in 16% (4/25) of the patients.

Transduction of the constructs in BaF3 cells revealed that none had transforming capacities. Overexpression of the wild type RTK could make the cells IL3 independent after stimulation of the kinase by its ligand.

Conclusion: The presence of a fusion transcript in 31% of the cell lines and 16% of patients with T-ALL suggests a possible role in the development of leukemia.