Detection of Immune Responses in Sentinel Nodes Draining Human Urinary Bladder Cancer

Abstracts

Scandinavian Society for Immunology35th Annual Meeting and 20th Summer School

Aarhus, Denmark, June 13–16, 2004

The following workshops will be running at the 35th Annual meeting of The Scandinavian Society forImmunology in Aarhus, Denmark, June 13–16, 2004. The number given to the abstracts in eachworkshop does not reflect the order of presentation.

Monday: AutoimmunityInfection and immunityStimulus and response

Tuesday: HypersensitivityCommensals and immunityComplement

Wednesday: TumourimmunologyImmunotechnology

Abstract Editors

Steffen ThielRalf Agger

Jesper ReinholdtHans Jurgen Hoffmann

# 2004 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 59, 609–637

AUTOIMMUNITY 1

Autoantibodies in Patients withRheumatoid Arthritis

A. Astrinidou-Vakaloudi, I. Diamanti, S. Xytsas,D. Xatzidimitriou & E. GeorgakopoulouDepartment of Microbiology, General Hospital of Thessaloniki ‘AgiosPavlos’, Thessaloniki, Greece. E-mail: [email protected]

Aim: The aim of this study is to examine the diagnosticvalue of autoanitbodies in patients suffering from rheuma-toid arthritis. We evaluated the presence of the followingautoantibodies: rheumatoid factor (RF), antinuclear anti-bodies (ANAs), antibodies against cadiolipin (a-CL) andantibodies against cyclic citrullinated peptide (anti-CCP).Methods: We studied the presence of RF, ANA, a-CL andanti-CCP in 40 patients with rheumatoid arthritis. Rheuma-toid factor was measured using nephelometric method, whileANAs were examined by indirect immunofluorescence tech-nique using Hep-2 cells as substrate. Sera that reacted at 1/80dilution were classified as ANA positive. Positive sera werestudied up to 1/1280 dilution. A-CL and anti-CCP weremeasured by enzyme-linked immunosorbent assay.Results: RF was positive in 30 patients (75%), ANA in 15(37%), a-CL in 10 (25%) and anti-CCP in 36 (90%).Predominant pattern of nuclear staining of ANA-positivesera was homogenous and speckled type. ANA titres wereparticularly low; most patients (6) had ANA titre equal to1/80, and five patients had a titre of 1/160, while only fourout of 40 had an ANA titre of 1/320.Conclusions: Autoimmune disorders such as RA arecharacterized by various autoantibodies that usually arenot specific, as they are present in many other diseases.However, RF and especially anti-CCP are very often andshow higher specificity for RA, being useful diagnosticserological markers. On the other hand, ANA and a-CLare less common in RA paitents; they may be useful interms of prognosis and treatment, but they always shouldbe evaluated in correlation with the clinical features andthe rest of the laboratory findings of each patient.

AUTOIMMUNITY 2

Plasma TNF-Binding Capacity and SolubleTNF Receptors in Patients with JuvenileIdiopathic Arthritis

B. Bjoernhart,1 P. Svenningsen,2

S. Gudbrandsdottir,1 M. Zak,2 S. Nielsen,2

K. Bentzen1 & K. Muller1,2

1Institute for Inflammation Research, Rigshospitalet, and 2PediatricDepartment, Rigshospitalet, Copenhagen, Denmark. E-mail:[email protected]

Background: Unbalanced production of proinflammatorycytokines may be related to disease progression in rheu-matoid arthritis and juvenile idiopathic arthritis (JIA).Within the TNF system, the two agonists, TNF-a andTNF-b, also called lymphotoxin-a (LT), are bound bysoluble TNF receptors (sTNFR-I and -II) that act asnatural inhibitors of TNF-induced inflammation. Weinvestigated the plasma levels of sTNFR-I in parallel withLT-binding capacity (LTBC) in patients with JIA.Methods: The levels of sTNFR-I were measured byELISA (R&D). LTBC was determined by spiking dilutedplasma samples with recombinant LT. Detectable LT wasmeasured by an in-house ELISA measuring unbound LTonly. LTBC was expressed in arbitrary units (AUs) as thepercentage value of bound LT to added LT.Result: In contrast to previous findings of elevatedsTNFR levels in patients with various chronic inflamma-tory diseases, we found slightly reduced sTNFR-I levels inJIA patients (n¼ 123) compared with age-matchedhealthy controls (n¼ 37): 1077 pg/ml (819–2280) versus1185 pg/ml (625–2303) [median (range)], P¼ 0015.However, the sTNFR-I levels correlated positively withthe number of active joints, physicians’ global assessmentand CRP. In contrast, patient LTBC values were elevatedcompared to healthy controls: 44 AU (36–52) versus31 AU (13–41), P< 0.0001.Conclusion: Despite overall slightly reduced plasma levelsof sTNFR-I, the capacity to bind TNF was increased inplasma samples from JIA patients. Studies to identify theTNF-binding substances in plasma are in progress.

AUTOIMMUNITY 3

Infliximab Treatment of RheumatoidArthritis Patients SimultaneouslyIncreases TNF-a Protein Levels andReduces mRNA Expression in the Blood

T. O. Hjelmevik,1 A. G. Kvalvik,2 P. M. Knappskog,1

L. Lefsaker,2 A. S. Kleiven,2 S. Dyvik,2 J. G. Brun,1

H. Østergaard & H. G. Eiken1

1Haukeland University Hospital, 2Haugesund Sanitetsforening Revmatis-mesykehus, and 3Department Laboratory Haugesund Hospital, Bergen,Norway. E-mail: [email protected]

Objective: Tumour necrosis factor-a (TNF-a) is animportant mediator in the pathogenesis of rheumatoidarthritis (RA). We have investigated long-term anti-TNF-a treatment with infliximab with respect to TNF-a geneactivity and protein levels in the blood of RA patients anddisease activity score (DAS).Methods: TNF-a mRNA and plasma protein in RApatients (n¼ 29) and healthy controls (n¼ 24) was deter-mined before and during treatment with infliximab (3mg/kg)

610 Abstracts..................................................................................................................................................................................................


using real-time quantitative reverse transcription-polymerasechain reaction (RT-PCR) and high sensitivity enzyme-linkedimmunosorbent assay (ELISA), respectively. The diseaseactivity of the patients was assessed as DAS value.Results: The TNF-a mRNA levels of RA patients at base-line were higher than that of the control group (P¼ 0.0135)but were significantly reduced after initiation of treatment(P< 0.001). Low mRNA levels were sustained throughoutthe 54 weeks of the study. Baseline protein levels of RApatients were similar to the control group. After 2 weeks oftreatment, the protein levels were significantly elevated frombaseline (P¼ 0.0353) and increased throughout week 14.Clinical improvement for all RA patients was found uponinfliximab treatment, as a reduction in DAS values(P< 0.001). The increase in protein and reduction in DASvalue from week 2–14 was also correlated (P¼ 0.0374).Conclusion: During infliximab treatment of RA patients,there is an accumulation in immune-reactive TNF-a pro-tein in blood plasma and simultaneously a reduction inTNF-a gene expression in PBMC, which may in partexplain the beneficial course of RA symptoms.

AUTO IMMUNITY 4

Anti-Inflammatory Liver X Receptors andRelated Molecules in Multiple SclerosisPatients from Sardinia and Sweden

Y.-M. Huang,1 X. Liu,1 K. Steffensen,2 A. Sanna,3

G. Arru,3 A. Sominanda,1 S. Sotgiu,3 G. Rosati,3

J.-A. Gustafsson2 & H. Link1

1Division of Neuroimmunology, Neurotec Department, Karolinska Institute,Stockholm, 2Department of Biosciences, Karolinska Institutet at NOVUM,Huddinge, Sweden, and 3Institute of Clinical Neurology, University ofSassari, Sassari, Italy. E-mail: [email protected]

The nuclear receptor heterodimers of liver X receptors(LXRs) are recently identified as key transcriptional regu-lators of genes involved in lipid homeostasis and inflam-mation. LXRs and their ligands are negative regulators ofmacrophage inflammatory gene expression. Multiplesclerosis (MS), a demyelinating disease of the central ner-vous system of unknown cause, is characterized by recur-rent inflammation involving macrophages and theirinflammatory mediators. Sweden belongs to the countrieswith a high MS incidence. In Italy, incidence is lower, withan exception for Sardinia where the incidence is evenhigher than that in Sweden. Subjects from Sardinia areethnically more homogeneous and differ from Swedes,also regarding genetic background and environment. Westudied LXRs and their related molecules of bloodmononuclear cells (MNCs) from female patients withuntreated relapsing-remitting MS from Sassari, Sardiniaand Stockholm, Sweden. Sex- and age-matched healthy

controls (HCs) were from both areas. mRNA expressionwas evaluated by real-time PCR. LXR-a was lower(P< 0.05) in MS (mean� SEM: 3.1� 0.2; n¼ 37) com-pared to HC (3.6� 0.1; n¼ 37). LXR-a was lower in MSfrom Stockholm (2.6� 0.2; n¼ 22) compared to corre-sponding HC (3.4� 0.1; n¼ 22; P< 0.01) and comparedto MS (3.8� 0.2; n¼ 15; P< 0.001) and HC (4� 0.2;n¼ 15; P< 0.001) from Sardinia. MS patients fromStockholm, but not from Sassari, also expressed lower(P< 0.05) LXR-b (�4.1� 0.4) compared to correspond-ing HC (�2.9� 0.3). MS from Stockholm was associatedwith higher ABCA-1 (6.1� 0.4 versus 5.0� 0.3;P< 0.05) and higher estrogen receptor-b-Cx (2.4� 0.4versus 0.8� 0.4; P< 0.01) compared to correspondingHC. The HC from Sassari had higher androgen receptor(2.9� 0.2) compared to MS from Sassari (1.4� 0.3;P< 0.01), MS (1.3� 0.4; P< 0.01) and HC fromStockholm (1.2� 0.3; P< 0.01). MS from Sassari hadlower cyclooxygenase-1 compared to corresponding HC(5.1� 0.4 versus 6.6� 0.3; P< 0.01) and lower prosta-glandin-E (�0.03� 0.5) compared to the HC (1.4� 0.5;P< 0.05) and MS (2.7� 0.4; P< 0.05) and HC fromStockholm (1.9� 0.4, P< 0.001). Our findings identifyLXRs and their related molecules as being involved in MSfrom Stockholm but not from Sassari, while sex hormonereceptors seem to be involved in MS in Sassari.

AUTO IMMUNITY 5

Multiple Sclerosis: IFN-b InducesCD123+BDCA2– Dendritic Cells thatProduce IL-6 and IL-10 and have NoEnhanced Type I Interferon Production

Y. M. Huang,1 S. Adikari,1 U. Bave,2 A. Sanna1,3 &G. Alm4

1Division of Neuroimmunology, Neurotec Department, Karolinska Institute,Stockholm, 2Department of Medical Sciences, Uppsala University, Uppsala,Sweden, 3Institute of Clinical Neurology, University of Sassari, Sassari,Italy, and 4Division of Veterinary Immunology and Virology, Departmentof Molecular Biosciences, Biomedical Center, Uppsala, Sweden. E-mail:[email protected]

IFN-b, an approved drug for multiple sclerosis (MS), acts ondendritic cell (DC) by suppressing their production of IL-12p40 and increasing IL-10. This results in Th2-biasedimmune responses. The nature of IFN-b-modulated DCremains elusive. Previously, we observed that IFN-b dosedependently induces expression of CD123, i.e. a classicalmarker for plasmacytoid DC, on human blood monocyte-derived myeloid DC. Such IFN-b-modulated DC producepredominantly IL-10 but are IL-12 deficient, with potentTh2 promotion. In the present study, we further characterizeIFN-b-modulated DC by using recently identified blood

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DC antigens (BDCA) and investigate their ability to produceType I IFN in response to virus stimulation. We show thatIFN-b induces development of CD123þ DC from humanblood monocytes, which coexpress BDCA4þ but are nega-tive for BDCA2–, a specific marker for plasmacytoid DC.Such IFN-b-modulated DC produce large amounts of IL-6and IL-10, but no IL-12p40 and have no enhanced IFN-band IFN-b production. The findings indicate that IFN-b-modulated DC represent a myeloid DC subset with dimin-ished CD11c, BDCA-1 and CD1a expression, having potentTh2-promoting function but lacking antiviral capacity.

AUTOIMMUNITY 6

Peripheral Blood T-Cell Responses toKeratin Peptides that Share Sequenceswith M Proteins are Largely Restricted toSkin-Homing CD8+ T Cells

A. Johnston, J. E. Gudjonsson, H. Sigmundsdottir,T. H. Love & H. ValdimarssonDepartment of Immunology, Landspitali-University Hospital, Reykjavik,Iceland. E-mail: [email protected]

The association of psoriasis with throat infections by strep-tococcus pyogenes suggests a potential antigenic target forthe T cells that are known to infiltrate dermis and epider-mis of psoriatic skin. Streptococcal M protein shares anextensive sequence homology with human epidermal kera-tins. Keratins 16 (K16) and 17 (K17) are mostly absentfrom uninvolved skin but are upregulated in psoriaticlesions. There is increasing evidence that CD8þ T cellsplay an important effector role in psoriasis and M protein-primed T cells may recognize these shared epitopes in skinvia molecular mimicry. To identify candidate epitopes,peptides with sequences from K17 were selected on thebasis of predicted binding to HLA-Cw6 and sequencesimilarities with M6 protein. Matched peptides from thesequence of M6 protein and a set of peptides with poorpredicted binding were also selected. Cw6þ individualswith psoriasis and Cw6þ healthy controls, having a familyhistory of psoriasis, were recruited. PBMCs were incubatedwith the peptide antigens. T-cell activation in the CD4þ,CD8þ and later the skin-homing cutaneous lymphocyte-associated antigen (CLA)-expressing subset of CD8þ Tcells was evaluated by CD69 expression and intracellularIFN-g accumulation using flow cytometry. We demon-strate that Cw6þ psoriasis patients had significant CD8þ

T-cell IFN-g responses to peptides from K17 and M6protein selected on the basis of sequence homology andpredicted HLA-Cw*0602 binding. These responses wereabout 10 times more frequent in the skin-homing cuta-neous lymphocyte-associated antigen-expressing (CLAþ)subset of CD8þ T cells. CD4þ T cells showed only

borderline responses. CD8þ T cells from Cw6þnonpsoriatic individuals responded to some M6 peptidesbut very rarely to K17 peptides, and this also applied tothe CLAþCD8þ subset. These findings indicate that psori-atic individuals have CD8þ T cells that recognize keratinself-antigens and that epitopes shared by streptococcal Mprotein and human keratin may be targets for the CD8þ Tcells that infiltrate psoriatic skin lesions.

AUTOIMMUNITY 7

Citrullinated Proteins in Arthritis; theirPresence in Joints and Effects onImmunogenicity

K. Lundberg,1 S. Nijenhuis,2 E. Vossenaar,2

W. J. Venrooij,2 L. Klareskog1 & H. E. Harris1

1Rheumatology Unit, Department of Medicine, Karolinska Institutet,Stockholm, Sweden, and 2Department of Biochemistry, University ofNijmegen, Nijmegen, The Netherlands. E-mail: [email protected]

Autoantibodies directed against citrulline-containing pro-teins have an impressive specificity of nearly 100% in RApatients and a suggestive involvement in the pathogenesis.The targeted epitopes are generated by a post-translationalmodification catalysed by the calcium-dependent enzymepeptidyl arginine deaminase that converts the positivelycharged arginine to polar but uncharged citrullin. The aimof this study was to analyse the presence of citrulline in thejoints at different time points of collagen-induced arthritisin DA rats by immunohistochemistry and to investigatehow immunogenicity and arthritogenicity was affected bycitrullination of rat serum albumin (RSA) and collagen typeII (CII). Our results indicate that citrulline could bedetected in joints of arthritic animals, first appearance atthe onset of disease and increasing as disease progressed intoa chronic state. Unimmunized animals or time points beforeclinical signs of arthritis were negative. By morphology, westate that some infiltrating macrophages as well as thecartilage surface stain positive for citrulline, while themajor source of citrullinated proteins appears to be fibrindepositions. A specific Cit-RSA T-cell response wasobserved in animals challenged by citrullinated RSA, noresponse was recorded when RSA was used as a stimulus.The IgG analysis reveals not only a response towards themodified protein but also cross-reactivity to native RSA. NoT-cell or B-cell response was noted in animals injected withunmodified RSA. Cit-CII induced a disease with higherincidence and earlier onset than did the native counterpart.We conclude that, in contrast to the human disease, citrul-line does not seem to appear before clinical signs. Asinflammation proceeds, citrulline is detected specifically inthe joints. All other organs investigated were negative. We

612 Abstracts..................................................................................................................................................................................................


also conclude that citrullination of a protein can breaktolerance and increase its arthritogenic properties.

AUTO IMMUNITY 8

Germinal Centres in Primary Sjogren’sSyndrome Indicate a Certain ClinicalImmunological Phenotype

M. V. Jonsson,1 J. G. Brun,2 K. Skarstein1 &R. Jonsson1,2,3

1Department of Oral Pathology, Institute of Odontology, University ofBergen, 2Department of Rheumatology, Haukeland University Hospital,3Broegelmann Research Laboratory, The Gade Institute, University ofBergen, and 4Department of Oto-Rhino-Laryngology/Head and NeckSurgery, Haukeland University Hospital, Bergen, Norway. E-mail: [email protected]

Ectopic germinal centers (GCs) can be detected in thesalivary glands of approximately 1/5 of patients withSjogren’s syndrome (SS) and appear in both primary andsecondary SS. Previously, ectopic GC have been associatedwith increased local autoantibody production. The aim ofthis study was to determine whether GC in primary Sjogren’ssyndrome (pSS) defines a distinct seroimmunological pheno-type. Retrospectively, a material of 130 haematoxylin andeosin-stained paraffin-embedded tissue sections of minorsalivary gland tissue from patients with pSS was morpholo-gically screened for the presence of ectopic GC. GC-likelesions were detected in 33/130 (25%) of the pSS patients.Seventy-two pSS patients lacking these structures (GC-) wererandomly selected for comparison. Focus score was signifi-cantly increased in the GCþ patients compared to the GC–

patients (P¼ 0.035). In the GCþ group, 54.5% of thepatients presented with anti-Ro/SSA compared to 43.7% inthe GC– group. Anti-La/SSB was detected in 31.3% of theGCþ patients compared to 25.7% of the GC– patients.Sixty-one percentage of GCþ patients presented withincreased levels of IgG, a nonsignificant difference whencompared to 39.4% in the GC– patients (P¼ 0.089). Levelsof RF, ANA, ENA, IgM and IgA were similar in both patientgroups, as were ESR and CRP. In conclusion, patients withectopic GC have a higher focus score and more often presentwith autoantibodies and increased levels of IgG comparedto pSS patients with regular focal infiltration (GC–). Ourfindings may indicate a certain seroimmunological phenotypeand warrant for further prospective studies.

AUTO IMMUNITY 9

Association between Mannose-BindingLectin and Vascular Complications in Type1 Diabetes

T. K. Hansen,1 L. Tarnow,2 S. Thiel,3 R. Steffensen,4

H.-H. Parving2 & A. Flyvbjerg1

1Immunoendocrine Research Unit, Medical Department M, Aarhus Uni-versity Hospital, Aarhus, 2Steno Diabetes Center, Gentofte, 3Department ofMedical Microbiology and Immunology, University of Aarhus, Aarhus, and4Regional Centre for Blood Transfusion and Clinical Immunology, AalborgHospital, Aalborg, Denmark. E-mail: [email protected]

Complement activation and inflammation have been sug-gested in the pathogenesis of diabetic vascular lesions. Weinvestigated serum mannose-binding lectin (MBL) levelsand polymorphisms in the MBL gene in type 1 diabetic(T1DM) patients with and without diabetic nephropathyand associated macrovascular complications. Polymorph-isms in the MBL gene and serum MBL levels were deter-mined in 199 T1DM patients with overt nephropathy and192 T1DM patients with persistent normoalbuminuriamatched for age, sex and duration of diabetes as wellas in 100 healthy control subjects. The frequencies ofhigh and low expression MBL genotypes were similar inpatients with T1DM and healthy controls. High MBLgenotypes were significantly more frequent in diabeticpatients with nephropathy than in the normoalbuminuricgroup, and the risk of having nephropathy, given ahigh MBL genotype, assessed by odds ratio was 1.52(1.02–2.27), P¼ 0.04. Median serumMBL concentrationswere significantly higher in patients with nephropathythan in patients with normoalbuminuria [2306 mg/l (IQR753–4867 mg/l) versus 1491 mg/l (IQR 577–2944),P¼ 0.0003], and even when comparing patients withidentical genotypes, serum MBL levels were higher in thenephropathy group than in the normoalbuminuric group.Patients with a history of cardiovascular disease had sig-nificantly elevated MBL levels independently of nephro-pathy status [3178mg/l (IQR 636–5231mg/l) versus 1741mg/l(IQR 656–3149mg/l), P¼ 0.02]. The differences in MBLlevels between patients with and without vascular complica-tions were driven primarily by pronounced differencesamong carriers of high MBL genotypes (P< 0.0001). Ourfindings suggest that MBL may be involved in the patho-genesis of microvascular and macrovascular complicationsin type 1 diabetes and that determination of MBL statusmight be used to identify patients at increased risk ofdeveloping these complications.

AUTO IMMUNITY 10

Protective DNA Vaccination AgainstMOG91-108-Induced ExperimentalAutoimmune Encephalomyelitis InvolvesInduction of IFNbJ. Wefer, R. A. Harris & A. Lobell

Abstracts 613..................................................................................................................................................................................................


Neuroimmunology Unit, Center for Molecular Medicine, Karolinska Insti-tutet, Stockholm, Sweden. E-mail: [email protected]

DNA vaccine coding for the encephalitogenic peptideMOG91-108 protects LEW.1AV1 from subsequent devel-opment of experimental autoimmune encephalomyelitis(EAE). Protection is associated with a type 1 immuneresponse and is dependent on the presence of CpG DNAmotifs. The mechanisms underlying the observed reduc-tion of EAE development in protected rats have not beenfully clarified. We investigated immunological characteris-tics of lymphocytes after DNA vaccinaton and subsequentEAE induction. We confirm that protection was not asso-ciated with suppression of T1 cells, as transcription of thenovel molecule rat T-cell immunoglobulin- and mucin-domain-containing molecule (TIM-3), reported to beexclusively expressed on differentiated T1 cells, was notaltered by DNA vaccination. We did not note any clonaldeletion upon tolerization, but detected an antigen-specificlymphocyte population upregulating IFNg upon recallstimulation 3 weeks after protective DNA vaccination. Inprotected rats, we observed (1) no alterations in antigen-specific Th2 or Th3 responses, (2) reduced MHC IIexpression on splenocytes early after EAE induction, (3)antigen-specific upregulation of IFNb upon recall stimula-tion and (4) reduced IL-12Rb2 on lymphocytes. Wethus demonstrate an association of the protective effectof DNA vaccination with expression of IFNb. We arecurrently investigating the cellular mechanisms behindthis IFNb-mediated protection.

AUTOIMMUNITY 11

The Role of Immune ComplexesConsisting of Myelin Basic Protein (MBP),Anti-MBP Antibodies and Complement inPromoting CD4+ T-cell Responses to MBPin Health and Multiple Sclerosis

C. J. Hedegaard, K. Bendtzen & C. H. NielsenInstitute for Inflammation Research, University Hospital, Copenhagen,Denmark. E-mail: [email protected]

Multiple sclerosis (MS) is an autoimmune condition char-acterized by degeneration of nerve fibre myelin sheets. Acandidate autoantigen, myelin basic protein (MBP), hasespecially attracted attention. The presence of anti-MBPantibodies is a predictor of definite MS, but their rolein the pathogenesis remains obscure. T cells have longbeen known to play a pivotal role in the pathogenesisof MS. Recently, an important role for B cells as auto-antigen-presenting cells has been demonstrated in otherautoimmune diseases, including rheumatoid arthritis anddiabetes. The uptake of MBP by B cells and the

presentation of MBP-derive peptides to T helper (Th)cells by B cells may be promoted by the formation ofcomplement (C) activating immune complexes (ICs)between MBP and natural autoantibodies in healthy indi-viduals and disease-associated anti-MBP antibodies in MSpatients, respectively. We have investigated the formationof MBP-containing IC, the binding of MBP to B cells, theMBP-elicited induction of Th-cell and B-cell proliferationand the cytokine production in peripheral blood mono-nuclear cells (PBMCs) from healthy donors grown in thepresence of intact or C-inactivated serum from healthydonors or patients with MS. While MBP did not inducemeasurable proliferation of B cells nor CD4þ T cells, weobserved the production of TNF-a, IFN-g and IL-10 byPBMC in response to incubation with MBP in the pre-sence of sera from healthy controls as well as sera from MSpatients. By contrast, no production of IL-2, IL-4 and IL-5was detected. We are currently investigating the capabilityof MS sera to promote the formation of MBP-containingIC and thereby enhance the cytokine responses, by virtueof elevated anti-MBP contents.

INFECT ION AND IMMUNITY 1

Delayed Elimination of the LCM Virusfrom Acid Sphingomyelinase-DeficientMice due to Reduced Expansion of Virus-Specific CD8+ T Lymphocytes

O. Utermohlen, U. Karow, N. Baschuk, J. Herz,T. T. Loegters & M. KronkeInstitute for Medical Microbiology, Immunology and Hygiene, MedicalCenter of the University of Cologne, Cologne, Germany. E-mail: [email protected]

The phagolysosomally localized acid sphingomyelinase(ASMase) activated by proinflammatory cytokines such asTNF and IFN-g generates the signalling molecule cera-mide which in turn results in the activation of proteaseslike cathepsin D. These characteristics of ASMase suggest apossible role of this molecule in the phagocytotic uptakeand phagosomal degradation processes of antigens or inantigen presentation. We show here that ASMase–/– micefail to eliminate the noncytopathic lymphocytic chorio-meningitis (LCM) virus as rapidly as littermate wildtypemice. Investigation of the immune response revealed areduced expansion of CD8þ T cells. The secretion ofIFN-g in response to contact with target cells as wellas the cytolytic activity of virus-specific CD8þ T cellswas severely impaired. Additionally, both phases ofthe LCM virus-specific DTH response, mediated byCD8þ and CD4þ T cells consecutively, were diminishedin ASMase–/– mice. However, the secondary memoryresponse of virus-specific CTL was not altered, and the

614 Abstracts..................................................................................................................................................................................................


virus was effectively controlled for at least 3 months byASMase–/– mice. In conclusion, the results of this studysuggest an involvement of the ASMase in the activation,expansion or maturation of virus-specific CD8þ T cellsduring the acute infection of mice with the LCM virus.


Novel Markers for Alternative Activationof Macrophages: Macrophage Galactose-Type C-Type Lectins 1 and 2

R. Van Den Bergh,1 G. H. Hassanzadeh,1 L. Brys,1

B. K. Dahal,1 J. Brandt,2 J. Grooten,3

F. Brombacher,4 G. Vanham,5 W. Noel,1 P. Bogaert,3

T. Boonefaes,3 A. Kindt,1 P. de Baetselier1 & G. Raes1

1Department of Molecular and Cellular Interactions, Flemish Interuniver-sity Institute for Biotechnology, Free University of Brussels, Brussels,2Department of Veterinary Medicine, Institute for Tropical Medicine,Antwerp, 3Department of Molecular Biomedical Research, Flemish Inter-university Institute for Biotechnology, Ghent University, Ghent, Belgium,4Department of Immunology, University of Cape Town, Groote SchuurHospital, Cape Town, South Africa, and 5Laboratory of Immunology,Department of Microbiology, Institute for Tropical Medicine, Antwerp,Belgium. E-mail: [email protected]

In parallel with the Th1/Th2 dichotomy, macrophages arecapable of developing into functionally and molecularlydistinct subpopulations, due to differences in, for examplecytokine environment and pathological conditions. Whilethe best-studied, classically activated macrophage isinduced by type I stimuli such as IFN-g, a type II cytokineenvironment antagonizes the classical activation of macro-phages and is capable of alternatively activating macro-phages. However, molecular markers associated withthese type II cytokine-dependent, alternatively activatedmacrophages remain scarce. Besides the earlier documen-ted markers macrophage mannose receptor and arginase 1,we recently demonstrated that murine alternatively acti-vated macrophages are characterized by increased expres-sion of FIZZ1 and Ym. We now report that expression ofthe two members of the mouse macrophage galactose-typeC-type lectin gene family, termed mMGL1 and mMGL2,is induced in diverse populations of alternatively activatedmacrophages, including peritoneal macrophages elicitedduring infection with the protozoan Trypanosoma brucei orthe Helminth Taenia crassiceps, and alveolar macrophageselicited in a mouse model of allergic asthma. We alsodemonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mMGL1 and mMGL2 expression andthat, in vivo, induction of mMGL1 and mMGL2 isdependent on interleukin-4 receptor signalling. Moreover,we show that regulation of MGL expression is similar inhuman monocytes and monocyte-derived macrophages.Hence, macrophage galactose-type C-type lectins represent

novel markers for both murine and human alternativelyactivated macrophages; thus, paving the way for furthercharacterization of the phenotype of macrophagesoccurring in Th2 conditions.


Mapping of the Ex Vivo Cellular ImmuneResponse Against the Complete HumanParvovirus B19 Genome During AcuteInfection

A. Isa, O. Norbeck, C. Pohlmann & T. TolfvenstamDivision of Clinical Virology, Karolinska Institutet, Karolinska UniversityHospital at Huddinge Hospital, Stockholm, Sweden. E-mail: [email protected]

Background: Human parvovirus B19 (B19) is a ubiqui-tous pathogen, normally causing a mild self-limiting dis-ease, but also capable of causing both significant pathologyand long-term persistence. The small size and stability ofthe virus makes it suitable for mapping of the full breathand the kinetics of the cellular immune responses follow-ing acute viral infection.Methods: Five patients with acute primary B19 infectionwere included in the study and followed consecutively forup to 200 weeks. Cellular immune responses were mappedby IFNg enzyme-linked immunospot to overlappingpeptides spanning the whole B19 genome.Results: In all five acutely infected patients, we were able tomonitor the kinetics of a strong specific cellular immunereaction. Responses peaked at levels of 850–1850 SFC/million PBMCs, roughly corresponding to 0.3–0.6% B19-specific CD8þ cells circulating in peripheral blood at 10–80weeks post-infection. The responses in individual patientswere directed to three or four different peptide pools, andthe specificity was confined to the same CD8 epitopespresent in the pools throughout the follow-up period. Themajority of responses were directed to the virus nonstruc-tural protein, only two patients showed any response to thecapsid proteins, elicited by the same epitope in both cases.Conclusion: The cellular immune responses to acute B19infection are surprisingly narrow in distribution andremain at high levels for up to 80 weeks post-infection.The initial epitope specificity is maintained, and themajority of responses target the virus nonstructural pro-tein, which is not included in vaccine preparations,evaluated against the infection.


Malaria and Nutritional Status inChildren Living on the Coast of Kenya

Abstracts 615..................................................................................................................................................................................................


A. M. Nyakeriga,1,2,3 M. Troye-Blomberg,2

A. K. Chemtai,3 K. Marsh1 & T. N. Williams1

1KEMRI/Wellcome Trust Programme, Centre for Geographic MedicineResearch, Coast, Kilifi District Hospital, Kilifi, Kenya, 2Stockholm University,Department of Immunology Wenner-Gren Institute, Stockholm, Sweden,and 3Moi University, Faculty of Health Sciences, Eldoret, Kenya.E-mail: [email protected]

The relationship between malnutrition and malaria is con-troversial. On one hand, malaria may cause malnutrition,while on the other, malnutrition itself may modulatesusceptibility to the disease. We investigated the associ-ation between Plasmodium falciparum malaria and malnu-trition in a cohort of children living on the coast of Kenya.The study involved longitudinal follow-up for clinicalmalaria episodes and anthropometric measurements atfour cross-sectional surveys. We used Poisson regressionanalysis to investigate the association between malaria andnutritional status. Compared to baseline (children with aWAZ or HAZ score of ��2), the crude incidence rateratios (IRRs) for malaria in children with low HAZ orWAZ scores (<�2) during the period prior to assessmentwere 1.17 (95% CI 0.91–1.50; 0¼ 0.21) and 0.94(0.71–1.25; 0.67), respectively, suggesting no associationbetween malaria and the subsequent development of PEM.However, we found that age was acting as an effect modi-fier in the association between malaria and malnutrition.The IRR for malaria in children 0–2 years old who weresubsequently characterized as wasted was 1.65 (1.10–2.20;P¼ 0.01), and a significant overall relationship betweenmalaria and low-HAZ was found on regression analysiswhen adjusting for the interaction with age (IRR 1.89;1.01–3.53; P< 0.05). Although children living on thecoast of Kenya continue to suffer clinical episodes ofuncomplicated malaria throughout their first decade, theassociation between malaria and malnutrition appears tobe limited to the first 2 years of life.


Presence of Helicobacter pyloriAntibodies in Haemodialysis Patients

A. Astrinidou-Vakaloudi,1 S. Xytsas,1 I. Diamanti,1

H. Ioannidis2 & P. Pangidis2

1Microbiology Department of General Hospital of Thessaloniki ‘AgiosPavlos’, Thessaloniki, Greece, and 2Nefrology, 2nd IKA Hospital ofThessaloniki, Thessaloniki, Greece. E-mail: [email protected]

Aim: Renal dysfunction may influence the colonization ofgastric mucosa by urea-splitting bacteria such as Helicobac-ter pylori, by increasing urea concentrations in the gastricjuice. Our aim was to investigate the prevalence ofH. pylori in patients with end-stage renal disease (ESRD),receiving long-term haemodialysis treatment.

Methods: This study included 40 sera from patients withESRD (29 male and 11 female) undergoing periodic hae-modialysis; mean time of treatment was 42.6 months.Using ELISA technique, we investigated the presence ofIgG and IgA antibodies against H. pylori as well as IgGCagA (antibodies specific for CagA(þ) strains of H.pylori). Sera from 40 healthy blood donors were used asa control group.Results: H. pylori IgG antibodies were detected in 32 outof 40 (80%) patients in the dialysis group, while 31/40(77.5%) tested positive for IgA. IgG CagA antibodies werepresent in 13 out of 40 (32.5%). Prevalence of H. pyloriIgG, IgA and CagA IgG antibodies in the control groupwas 33, 7 and 15%, respectively.Conclusions: Although international data suggest thatprevalence of H. pylori infection is the same in ESRDpatients as in healthy individuals, in our study that seemsnot to be the case. The higher blood and gastric juice urealevels may be a risk factor (among many others), but morestudies are required in order to understand the relation ofH. pylori infection in this group of patients.


Mucosally Targeted Prime-BoostVaccination Approaches for TuberculosisBased on the TLR2/4 Ligand OprIAdjuvant

T. Gartner,1 M. Baeten,1 P. De Baetselier,1

K. Huygen2 & H. Revets1

1Flanders Interuniversity Institute for Biotechnology, Department ofMolecular and Cellular Interactions, Free University of Brussels, Brussels,and 2Pasteur Institute of Brussels, Mycobacterial Immunology, Brussels,Belgium. E-mail: [email protected]

Immunity against tuberculosis (TB), caused by Mycobac-terium tuberculosis, depends largely on activation andmaintenance of strong cell-mediated immune responsesinvolving both CD4þ and CD8þ T cells and the abilityto respond with Th1-type cytokines, particularly IFN-g.Recent studies suggested that BCG, the only licensedvaccine against M. tuberculosis, may fail to induce T-cellresponses in the lung mucosa and may therefore not pro-tect against pulmonary TB. A decrease in TB mortalitymay be achieved by enhancing immunity in the lung. Thepresent study evaluated the induction of antigen-specificimmunity in the lung by intranasal (i.n.) delivery of thelipoprotein I (OprI) from Pseudomonas aeruginosa. OprIhas shown to be a Toll-like receptor 2/4 agonist that, whengiven subcutaneously, induces Type-1 immune responsesagainst heterologous antigens. Here, a fusion of OprIto Ag85A of Mtb (OprI-Ag85A) was used as a subunitvaccine in homologous prime-boost immunizations. In

616 Abstracts..................................................................................................................................................................................................


addition, OprI-Ag85A was combined with an Ag85A-encod-ing DNA vaccine (Ag85A DNA) or with BCG in hetero-logous prime-boost vaccinations. Intranasal and parenteraldelivery with OprI-Ag85A elicited comparable T-cellresponses in the spleen; in addition, i.n. delivery elicitedspecific T-cell responses in the lung lymph nodes (LLNs).Intramuscular delivery of Ag85A DNA induced significantsystemic Th1 immune responses. Intranasal boosting withOprI-Ag85A enhanced this response and in addition inducedan antigen-specific IFN-g response in LLN. OprI may there-fore be an efficient adjuvant for mucosal boosting. We con-tinue to evaluate the protection induced by OprI-basedprime-boost vaccinations against pulmonary TB. Results onthe immunogenicity and protection against intravenous MtbH37Rv infection will be presented.


Differential Requirements for Toll-LikeReceptor Signalling for Induction ofChemokine Expression by Herpes SimplexVirus and Sendai Virus

J. Melchjorsen,1 A. G. Bowie,2 S. Matikainen3 &S. R. Paludan1

1Department of Medical Microbiology and Immunology, University ofAarhus, Aarhus, Denmark, 2Department of Biochemistry, Trinity College,Dublin, Ireland, and 3Department of Microbiology, National Public HealthInstitute, Helsinki, Finland. E-mail: [email protected]

Toll-like receptors (TLRs) are pattern recognition recep-tors of the innate immune system, which recognize molecu-lar structures on pathogens or cellular stress-associatedmolecules. TLR–ligand interactions trigger activation ofinflammatory signal transduction and expression of genesinvolved in host defense. In this study, we have examinedthe requirement for different TLR adaptor molecules invirus-induced chemokine expression and are currently tryingto identify the TLR involved. We have found that both aherpesvirus [herpes simplex virus (HSV)] and a paramyxo-virus (Sendai virus) require a functional genome to induceexpression or proinflammatory chemokines in human andmurine monocytic cell lines. For both viruses, this is inde-pendent of the TLR adaptor molecules TRIF and Mal.However, overexpression of the Vaccinia virus-encoded inhi-bitor of TLR-signalling A52R or dominant-negative MyD88totally inhibited HSV-induced RANTES expression butonly partially prevented Sendai virus from inducing thischemokine. This suggests that HSV-induced RANTESexpression occurs via a TLR pathways, whereas Sendaivirus utilizes both TLR-dependent and -independent path-ways to stimulate expression of RANTES. We are currentlytrying to identify the TLRs involved. Data from these studieswill also be presented at the meeting.


Crystal Structure of the 20-Specific andDouble-Stranded RNA-ActivatedInterferon-Induced Antiviral Protein 20-50-Oligoadenylate Synthetase

R. Hartmann,1,2 J. Justesen,1 S. Sarkar,2 G. Sen2 &V. Yee3

1Department of Molecular Biology, University of Aarhus, Aarhus, Denmark,2Department of Molecular Biology, The Cleveland Clinic Foundation, and3Department of Biochemistry, Case Western Reserve University, Cleveland,OH, USA. E-mail: [email protected]

20-50-oligoadenylate synthetases are interferon-induced,double-stranded RNA-activated antiviral enzymes whichare the only proteins known to catalyse 20-specificnucleotidyl transfer. This first crystal structure of a 20-50-oligoadenylate synthetase reveals a structural conservationwith the 30-specific poly(A) polymerase that, coupledwith structure-guided mutagenesis, supports a conservedcatalytic mechanism for the 20- and 30-specific nucleotidyltransferases. Comparison with structures of other super-family members indicates that the donor substrates arebound by conserved active site features while the acceptorsubstrates are oriented by nonconserved regions. The 20-50-oligoadenylate synthetases are activated by viral double-stranded RNA in infected cells and initiate a cellularresponse by synthesizing 20-50-oligoadenylates, that in turnactivate RNase L. This crystal structure suggests thatactivation involves a domain–domain shift and identifiesa putative dsRNA activation site that is probed bymutagenesis. We demonstrated that this site is requiredboth for the binding of dsRNA and for the subsequentactivation of OAS. This RNA-binding site is differentfrom known RNA-binding site; rather than forming adefined three-dimensional domain, it is located at theinterface of the two major domains in OAS. This novelarchitecture ensures that the dsRNA helix can makesimultaneously contact with both domains of OAS andensure the subsequent structural rearrangement leading tothe activation of OAS. Our work provides structuralinsight into cellular recognition of double-stranded RNAof viral origin and identifies a novel RNA-binding motif.


Pneumococcal IgA1 Protease ActivityInterferes with Opsonophagocytosis ofStreptococcus Pneumoniae Mediated bySerotype-Specific Human MonoclonalIgA1 Antibodies

J. Reinholdt,1 H. Baxendale,2 N. Ekstrom,3

Abstracts 617..................................................................................................................................................................................................


H. Kayhty,3 K. Poulsen1 & M. Kilian1

1Department of Medical Microbiology and Immunology, University ofAarhus, Aarhus, Denmark, 2Institute for Child Health, London, UK, and3National Public Health Institute (KTL), Helsinki, Finland. E-mail:[email protected]

Bacteria-specific IgA antibodies are efficient opsonins forneutrophils and mononuclear phagocytes, provided thatthe phagocytes express the Fca receptor (CD89). Expres-sion of CD89 can be stimulated by inflammatory cyto-kines, activated complement factors and certain microbialcomponents. In one study, unstimulated phagocytes wereable to ingest IgA antibody-treated pneumococci, but onlyin the presence of complement, which was found to beactivated by the IgA antibodies along the alternative path-way. Pneumococci produce IgA1 protease that cleaveshuman IgA1, but not IgA2, molecules in the hinge region.This leaves IgA1 as Faba (monovalent) deprived of Fcawhich contains the docking site for CD89. IgA1 is thevastly predominant subclass of IgA in the upper airwaysand circulation of humans.Aims: To examine the effects of IgA1 protease activity andcomplement on phagocytosis of IgA antibody-coatedpneumococci by an unstimulated human phagocytic cellline (hl60).Materials and methods: IgA1 and IgA2 monoclonal anti-bodies to serotype 4 pneumococcal capsular polysaccharide(ps) were generated by heterohybridoma technique invol-ving B cells from human vaccinees. Isogenic serotype 4pneumococci with and without IgA1 protease activity,respectively, were obtained after inactivation of the igagene of the TIGR4 strain. Opsonophagocytosis was quan-titated using the assay described by Romero-Steiner et al.Based on enumeration of surviving bacteria by culture.The integrity of IgA molecules was examined by westernblotting.Results: Both IgA1 and IgA2 antibody to type-4 polysac-charide-induced phagocytosis of IgA1 protease-deficienttype-4 pneumococci equally well in the absence as in thepresence of complement. Iga1 antibody to type-4 polysac-charide displayed a fourfold higher opsonophagocytosistiter against IgA1 protease deficient compared to homo-logous wildtype target bacteria. A similar effect of IgA1protease activity of the target bacteria was not observed ina parallel experiment where IgA2 antibody to type-4 poly-saccharide served as opsonin. IgA1 antibody extractedfrom IgA1 protease-producing target bacteria was almostexclusively in the form of Faba. Conversely, IgA1 fromprotease-deficient bacteria and IgA2 from both types ofbacteria were intact.Conclusions: These results indicate that the IgA1 proteaseactivity of S. neumoniae may help the bacteria escape IgA1antibody-mediated opsonophagocytosis. Besides, in theseexperiments, IgA-mediated opsonophagocytosis was inde-pendent of complement.

ST IMULUS AND RESPONSE 1

Supplementation of Vitamin C to WeanerDiets Increases IgM Concentration andImproves the Biological Activity ofVitamin E in Alveolar Macrophages

C. Lauridsen & S. K. JensenDepartment of Animal Nutrition and Physiology, Research CentreFoulum, Danish Institute of Agricultural Sciences, Foulum, Denmark.E-mail: [email protected]

Vitamins E and C have been found to increase the cellularand humeral immunity of pigs. Vitamin E deficiencyhas also been found to predispose pigs to different diseases,E. coli infection is one among them. After weaning, thevitamin E status of pigs often decreases to a critical lowlevel. In this experiment, we studied whether vitamin Csupplementation would be a possible feeding strategy tooptimize the immune status of weaners. The interactionbetween vitamin E and C is interesting due to the reportedsparing action on vitamin E or synergism between these tovitamins. Piglets were weaned at day 28 of age from sowsfed increasing dietary vitamin E during lactation, andpiglets were during the following 3 weeks fed either acontrol diet or this diet supplemented with 500mgSTAY-C per kg. Blood sampling was obtained weeklyfrom day 28 and until day 49 of age. On the same days,one piglet per dietary treatment was killed and alveolarmacrophages (AM) were harvested. Vitamin C supplemen-tation increased the concentration of IgM in serum ofpiglets throughout the weaning period. Although the vita-min E concentration in AM decreased with increasing ageof the piglets, the concentration was numerically higher inpiglets of sows fed the high dietary level of vitamin E.However, vitamin C supplementation tended to increasethe total AM concentration of vitamin E after weaning andincreased the proportion of the biologically most activeisomer of vitamin E [RRR-(a-tocopherol)] in the AM.The eicosanoid synthesis by AM was not influenced bythe vitamin C supplementation, but the synthesis ofleukotriene B4 was decreased 2 weeks after weaning com-pared to other days of AM harvesting. In conclusion,dietary vitamin C supplementation improved the immuneresponses of piglets after weaning.


Do High and Low Tumour NecrosisFactor-a Responders Exist in Dairy Cows?

C. M. Røntved,1 J. Dernfalk2 & K. L. Ingvartsen1

1Department of Animal Health and Welfare, Section of Production Dis-eases and Immunology, Ruminants, Danish Institute of AgriculturalScience, Tjele, Denmark, and 2Department of Anatomy and Physiology,

618 Abstracts..................................................................................................................................................................................................


Swedish University of Agricultural Sciences, Uppsala, Sweden.E-mail: [email protected]

A whole blood stimulation assay with Escherichia coli(O111:B4) endotoxin was established to measure the capa-city of dairy cows to produce the proinflammatory cyto-kine tumour necrosis factor-a (TNF-a) ex vivo. Initially, atime- and dose-dependent study was carried out to findthe optimal stimulation conditions for the TNF-aresponse. The TNF-a response peaked between 3 and4 h at 38.5 �C. A dose in the range of 5–10 g of E. colilipopolysaccharide (LPS)/ml whole blood was foundto give the maximum TNF-a response. Thirty-eightDanish–Holstein dairy cows were investigated for theirTNF-a responsiveness ex vivo in the periparturient period.Heparin-stabilized blood samples were collected seventimes over a period of 4 months (weeks �3, �1, 2, 3, 5,9 and 13 around calving) and stimulated with 5 g/ml of E.coli LPS. Indeed, fluctuations in the TNF-a responsivenessoccurred over time. Moreover, the mean TNF-a respon-siveness of 38 cows was found to be significantly increased(P< 0.001) in the weeks close to calving. However, in themore stabile physiological periods, some cows had a con-sistently low TNF-a response, whereas others had high aTNF-a response. We are currently investigating whetherhigh and low TNF-a responders to E. coli LPS also exist indairy cows in vivo. Moreover, the importance of TNF-aresponsiveness ex vivo to dairy cows’ susceptibility andclinical response to experimental E. coli infections in theudder is being investigated.


The Invertebrate Defence MoleculeCoelomic Cytolytic Factor, a FunctionalAnalog of the Cytokine Tumour NecrosisFactor-a, Interacts with Mammalian Cellsthrough its Lectin-Like Domain

R. Van Den Bergh,1 M. Bilej,2 P. de Baetselier1 &A. Beschin1

1Department of Molecular and Cellular Interactions, Flemish Interuniver-sity Institute for Biotechnology, Free University of Brussels, Brussels,Belgium, and 2Department of Immunology, Institute of Microbiology,Academy of Sciences of the Czech Republic, Prague, Czech Republic.E-mail: [email protected]

Coelomic cytolytic factor (CCF) is a 42 kDa invertebratepattern recognition molecule isolated from the coelomicfluid of the earthworm Eisenia foetida (Oligochaeta,Annelida). CCF displays a number of similarities withthe mammalian cytokine tumour necrosis factor-a (TNF-a) as a result of a shared N,N0-diacetylchitobiose lectin-likedomain. However, these similarities are solely functional

and are not based on any (DNA or amino acid) sequencehomology, thus suggesting a form of convergent evolution.In particular, the lectin-like domain of TNF-a has beenshown to induce membrane depolarization in various mam-malian cell types, through interactions with endogenousamiloride-sensitive ion channels. This nonreceptor-mediatedactivity of TNF-a has been reported to be involved in theresorption of oedema. Likewise, the lectin-like domain ofCCF also induces membrane depolarization in mammaliancells. Here, we show that CCF appears to be able to induceoedema resorption in an alveolar epithelial cell line throughits lectin-like domain. This lectin-like domain of CCF inter-acts (directly or indirectly) with endogenous sodium and/orchloride channels, and not potassium channels, on mamma-lian cells. Additionally, we suggest that the JNK/SAPK andErk1/2 pathways are involved in CCF-induced macrophageactivation. These results further establish the functionalanalogy between an invertebrate pattern recognition mole-cule and a mammalian cytokine and, from a more appliedpoint of view, suggest the possibility of utilizing CCF in thetreatment of oedema.


Release of sVEGF and sVEGFR1 fromWhite Blood Cells and Platelets DuringSurgery and Stimulation with BacterialAntigens

M. N. Svendsen,1 K. Werther,1 T. Bisgaard,1

I. J. Christensen2 & H. J. Nielsen1

1Department of Surgical Gastroenterology, Hvidovre University Hospital,and 2The Finsen Laboratory, National University Hospital, Copenhagen,Denmark. E-mail: [email protected]

Introduction: The influence of surgery on release of solu-ble vascular endothelial growth factor (sVEGF) and thesoluble vascular endothelial growth factor inhibitory recep-tor 1 (sVEGFR1) is unknown. We studied the effect ofmajor and minor surgery on potential variations in sVEGFand sVEGFR1 concentrations in vivo and on bacterialantigen-induced release of sVEGF and sVEGFR1 fromwhole blood in vitro.Methods: Sixty-one patients with abdominal diseasesundergoing five different surgical procedures wereincluded. Blood samples were drawn from anaesthetizedpatients before and after the operation. White blood cellsand platelets were counted, and plasma sVEGF andsVEGFR1 was determined by an ELISA method. Wholeblood from each blood sample was stimulated in vitro withbacteria-derived antigens (LPS or protein-A) and sVEGFand sVEGFR1 levels were subsequently determined in thesupernatants. Stimulation with isotonic saline served ascontrol assay.

Abstracts 619..................................................................................................................................................................................................


Results: Neither sVEGF or sVEGFR1 in plasma changedduring surgery. In vitro stimulation of blood samples withbacteria-derived antigens resulted in a significant increasein sVEGF (P< 0.0001) and a less pronounced but stillsignificant increase in sVEGFR1. Release of sVEGF due tostimulation was significantly higher after the operation(nonsignificant), whereas sVEGFR1 release remained lar-gely unchanged after surgery. Correlation between bacter-ial antigen-induced release of sVEGF and neutrophile cellcount was highly significant (P< 0.0001). There was nocorrelation between sVEGF and platelet cell count, andbacterial antigen-induced sVEGFR1 release did not corre-late with counts of neutrophils and platelets.Conclusions: Plasma sVEGF and sVEGFR1 concentrationsdid not change during surgery. In vitro bacterial stimulationled to increased release of sVEGF and sVEGFR1, whichwas not significantly amplified during surgery and whichmay be related to number of circulating neutrophils.


Natural Killer Cell Functions and SubsetsAfter In Vitro Stimulation with IL-2 andIL-12, with Special Emphasis onIntracellular IFN-g and NK-CellCytotoxicity

R. Nyboe,1,2 T. Rix,1,2 J. Krog,1,2 E. Tønnesen1 &M. Hokland2

1Department of Anaesthesiology and Intensive Care, Aarhus Universityhospital, and 2Institute of Medical Microbiology, and Immunology,University of Aarhus, Aarhus, Denmark. E-mail: [email protected]

Materials and methods: Isolated cryopreserved humanperipheral blood mononuclear cells (PBMCs) were stimu-lated with IL-2 and IL-12. This stimulation has previouslybeen shown to activate NK cells. Cell cytotoxicity wasmeasured by flow cytometry after incubation with k562cells. This method was compared to the current standard51Cr release assay. Cells were treated with BFA to accu-mulate IFN-g, stained for surface markers, permeabilizedand stained for intracellular IFN-g. Flow cytometry wasthen performed to measure intracellular IFN-g productionin PBMC, especially in NK cells.Results: We have demonstrated that stimulation with IL-2and IL-12 is effective in increasing the number of IFN-g-positive cells. There is a distinct difference between theCD3-CD56dim and the CD3-CD56bright subsets, with amuch greater proportion of IFN-g-positive cells in the CD3-CD56bright subset. The effects of stimulation with IL-2 andIL-12 on cytotoxicity will be presented, as will the relationbetween IFN-g production and cytotoxicity. In addition, wewill present results of these assays applied to porcine cells.

Discussion: In combination, these tests will address NKcell function by combining cytotoxicity with IFN-g pro-duction in NK cell subsets. The results will demonstratewhether this could serve as a useful tool in describing NK-cell function, which could be of value in clinical andexperimental settings.


Culture of Regulatory T-Cell Lines fromBronchial Mucosa

J. L. Rømer,1 J. Kelsen,2 R. Dahl1 &H. J. Hoffmann1

1Department of Respiratory Medicine, and 2Department of Gastroenter-ology, Aarhus University Hospital, Nørrebrogade, Arhus, Denmark. E-mail:[email protected]

T lymphocytes play a major role in many immuneresponses. In the last decade, special focus has been onthe function of Th1 and Th2 effector cells. Now theimportance of regulatory CD4þCD25þ T cells in main-tenance of the immunological homeostasis emerges. Sar-coidosis is a multisystem granulomatous disorder oftenaffecting the lungs. The typical sarcoid granulomas con-sists of epitheloid cells, macrophages and lymphocytes,mainly CD4þ T cells of Th1 phenotype. We have culturedT cells from bronchial biopsies of patients with sarcoidosisas well as from controls in high levels of interleukin 2 (IL-2) and IL-4 and demonstrate spontaneously arising CD4þ

CD25þ populations and high concentrations of IL-10 inthese cultures. The main difference between cultures ofsarcoid origin compared to controls is a very much higherconcentration of the inflammatory cytokines IL-6 andTNF-a in cultures of sarcoid origin.


The Effects of Hyperbaric Exposure onHuman Peripheral Blood MononuclearCells, with Special Emphasis on NaturalKiller Cell Cytotoxicity and Subsets

J. Krog,1,4 C. F. Jepsen,2 E. Tønnesen,1 E. Parner3

& M. Hokland4

1Department of Anaesthesiology and Intensive Care Medicine, AarhusUniversity hospital, 2Department of Orthopaedic Surgery, RandersCentralsygehus, 3Department of Biostatistics, 4Department of MedicalMicrobiology, and Immunology, The Bartholin Building, University ofAarhus, Aarhus, Denmark. E-mail: [email protected]

Materials and methods: As an experimental physio-logical stress model, we examined the effects of hyperbaricexposure on peripheral blood mononuclear cells (PBMCs)

620 Abstracts..................................................................................................................................................................................................


obtained from venous blood drawn from eight divers duringa simulated heliox saturation dive. Eight persons working innormobar atmosphere outside the pressurized chamberserved as control donors. The spontaneous cytotoxicity ofthe PBMCs was estimated in a 4 h 51Cr-release assay usingk562 as NK-sensitive target cells. The PBMCs werecharacterized, using 4-colour flow cytometry, with specialemphasis on the NK-cell subsets. The data were statisticallyanalysed using a multivariate regression model (Stata 8.2).P values <0.05 was considered statistically significant.Results: The estimated cytotoxicity increased significantlyin both the group of divers and control donors during thedive (pdivers< 0.01 and pcontrols< 0.01). Although thecytotoxicity increased relatively more (P< 0.01) in thegroup of divers compared to the group of control donorsbetween day 1 and 2.Discussion: The increased cytotoxicity of PBMC estimatedin the group of divers indicate that parts of the cellularimmune system are affected during the extreme physiologi-cal conditions induced during the initial phase of the pre-sented experimental hyperbaric setup. The increase incytotoxicity observed in the group of control donors couldhypothetically reflect the stress level in persons workingoutside the pressurized chamber during the dive.


Differential Effects of Interleukin-12 andInterleukin-10 on Superantigen-InducedExpression of Cutaneous Lymphocyte-Associated Antigen and aEb7 Integrin(CD103) by CD8+ T cells

H. Sigmundsdottir, A. Johnston, J. E. Gudjonsson& H. ValdimarssonDepartment of Immunology, Landspitali University Hospital, Reykjavik, Iceland.E-mail: [email protected]

The interaction with adhesion molecules expressed byvascular endothelium is the first step in lymphocyte infil-tration into tissues. At both cutaneous and mucosal sitesinterleukin-10 (IL-10), IL-12 and transforming growthfactor (TGF)-b are important regulators of chronicinflammatory disease, where cutaneous lymphocyte-associated antigen (CLA) and aE integrin (CD103) maybe expressed. Unlike CLA, CD103 is not believed to playa role in tissue-specific homing but may help to retainT cells within epithelial layers. We have previously shownthat IL-12 alone can together with an unknown cofactorincrease the expression of CLA. Stimulation with strepto-coccal pyrogenic exotoxin C (SpeC) increased the expres-sion of CD103 by CD8þ but not CD4þ T cells. WhileIL-12 increased superantigen-stimulated expression ofCLA, this cytokine strongly inhibited the CD103 expres-

sion, and a combination of IL-12 and TGF-b completelyabrogated the induced CD103 expression. Conversely,IL-10 suppressed CLA but increased CD103 expression.

These findings indicate that, in addition to suppressingthe development of Th1-mediated inflammatoryresponses, IL-10 may also inhibit the migration of CD8þ

T cells into the skin while IL-12 promotes such migration.Thus, the expression of CLA and CD103 may be antag-onistically regulated by IL-10 and IL-12, and the balancebetween these cytokines could influence the T-cell migra-tion of inflammatory cells into epithelial tissues.

HYPERSENS IT IV ITY 1

Proliferation of Cells in the Oral Mucosa,the Ear Skin and the Regional LymphNodes in Mice Sensitized and Elicitedwith a Hapten

E. Ahlfors,1 M. M. Sveinhaug,2 G. Nango,2

C. Johansen2 & T. Lyberg3

1Department of Oral Pathology and Forensic Odontology, Dental Faculty,2Dental Faculty, University of Oslo, and 3Research Forum, UllevaalUniversity Hospital, Oslo, Norway. E-mail: [email protected]

During contact sensitivity reaction, immune cells prolifer-ate. In order to study the histological picture of theseproliferation phases, we used a mouse model of contactsensitivity in the oral mucosa and on skin. We also usedbromodeoxyuridin (BrdU, an analogue to thymidin) thatis incorporated into the nucleus during cell replication.The hapten oxazolone (OXA) was used to sensitize andelicit the oral mucosa and/or the ear skin. Mice were killedat various times after elicitation, and unsensitized animalswere also exposed to the hapten as controls. BrdU (25mg/kg animal) was injected i.p. 2 h before the kill. Specimensfrom the oral mucosa, ear skin and submandibular andauricular lymph nodes were cut and fixed in 4% parafor-maldehyde. They were then treated with acid and biotiny-lated anti-BrdU antibody and developed using ABC-kitand DAB. The analyses were performed using a Leica lightmicroscope and the computer program ANALYSIS. In theoral mucosa, the frequency of proliferating cells wereincreasing during the observation period, 4–24 h afterelicitation, regardless of site of sensitization. The prolifer-ating cells were found mainly in the basal cell layer of theepithelium. Similar patterns were found in ear skin. Theregional lymph nodes demonstrated a few scattered pro-liferating cells 4 h after elicitation. After 24 h, these cellswere found frequently in the whole lymph node. Controlanimals exhibited considerable less proliferating cells at alltimes. We conclude that most proliferating cells werefound 24 h after elicitation locally at the hapten-exposedsites (the oral mucosa or the ear skin) as well as in theregional lymph nodes.

Abstracts 621..................................................................................................................................................................................................



Adenosine Receptor A2a is DifferentiallyExpressed in CD4þ T Lymphocytes ofAsthmatic and Healthy Individuals

P. Holopainen,1 C. Karagiannidis,1 B. Ruckert,1

G. Hense,2 C. Schmidt-Weber1 & K. Blaser1

1Swiss Institute of Allergy and Asthma Research, Davos, and 2Hochge-birgsklinik Davos Wolfgang, Davos-Wolfgang, Switzerland. E-mail:[email protected]

The endogenous nucleoside adenosine is released in excessduring inflammation or other metabolic stress and is gen-erally known to deliver tissue protective anti-inflammatoryeffects. Adenosine acts via four adenosine receptors ofwhich the A2a receptor is the predominant form in Tcells. Adenosine levels are elevated in asthmatic lung, andadenosine can directly induce mast cell degranulation andbronchoconstriction in these patients. Instead, the role ofanti-inflammatory mechanisms of adenosine on T cells inasthma is unclear.Aim: To study the A2a receptor expression in peripheralblood CD4þ T cells in asthmatic and healthy individualsusing flow cytometric and quantitative real-time PCRmethods.Results: Unstimulated CD4þ cells of asthmatic patientsexpressed significantly lower levels (P< 0.001) of A2areceptor in protein level (mean percentage of cells posi-tive� SEM: 76.8� 1.2, n¼ 6) compared to healthy indi-viduals (90.4%� 1.9, n¼ 4). Double staining forCD69 expression showed that stimulation of CD4þ cellsdecreased A2a expression in both groups but indicated thatthe detected lower levels of A2a in unstimulated cells ofasthmatics was not due to preactivation in these patients.Surprisingly, A2a mRNA expression in unstimulatedCD4þ cells was significantly higher (P< 0.05) in asth-matics (n¼ 28) compared to healthy controls (n¼ 7).The expression did not correlate with serum total IgElevels.Conclusions: Asthmatic individuals express less A2a ade-nosine receptor on their peripheral CD4þ T cells. Thehigher mRNA levels instead may point to a negative feed-back regulation in the receptor expression. The role ofpossibly decreased adenosine-mediated anti-inflammatoryeffects in asthma pathogenesis require further studies onthis T-cell mediated disease.


The Malassezia sympodialis Allergen Malas 11 with Sequence Similarity toManganese Superoxide DismutaseInduces Maturation and Production of

Inflammatory Cytokines in HumanDendritic Cells

M.Vilhelmsson, G. J. Ekman, A. Zargari &A. ScheyniusDepartment of Medicine, Clinical Allergy Research Unit, KarolinskaInstitutet, and Karolinska University Hospital, Stockholm, Sweden.E-mail: [email protected]

The chronic inflammatory skin disease atopic eczema (AE)affects almost 15% of the population in many countriestoday. The pathogenesis of AE is not fully understood. Acombination of genetic predisposition and environmentalfactors like microorganisms seems to contribute to thesymptoms. The yeast Malassezia sympodialis is part of ournormal skin micro flora but can act as an allergen and elicitspecific IgE and T-cell reactivity in patients with AE.Recently, we identified a novel major M. sympodialis aller-gen, designated Mala s 11 (22.4 kDa), with sequencesimilarity to the mitochondrial enzyme manganese super-oxide dismutase (MnSOD). Interestingly, Mala s 11 has ahigh degree of homology to human MnSOD. The aim ofthis study was to examine the effects of recombinant Malas 11 on antigen-presenting dendritic cells. Monocyte-derived dendritic cells (MDDCs) from healthy blooddonors were cultured with or without Mala s 11 fordifferent time periods. It was found that the maturationmarker CD83 and the costimulatory molecules CD80 andCD86 were upregulated on the MDDCs exposed to Malas 11 for 24 h, as demonstrated by flow cytometry. Further-more, coculture of MDDCs with Mala s 11 for 9 hinduced an increased production of the inflammatorycytokines IL-6 (200-fold), TNF-a (100-fold) and IL-8(sixfold), as detected by the cytometric bead array (CBA)analysis. Our results suggest that Mala s 11 affects theimmune response through DC maturation and productionof inflammatory cytokines. The potential cross-reactivitywith human MnSOD needs to be explored and the exactrole of Mala s 11 in the pathogenesis of AE assessed inclinical studies involving skin prick and atopy patch tests.


A Novel Adjuvant Allergen Complex,CBP-Fel d 1, Induces Upregulation ofCD86 and Cytokine Release in HumanDendritic Cells

T. Neimert-Andersson,1,2 G. J. Ekman,2

H. Gronlund,1 E. Buentke,2 A. Scheynius,2 M. VanHage-Hamsten1 & G. Gafvelin1

1Department of Medicine, Clinical Immunology and Allergy Unit,and 2Clinical Allergy Research Unit, Karolinska Institutet and

622 Abstracts..................................................................................................................................................................................................


Karolinska University Hospital, Stockholm, Sweden. E-mail:[email protected]

Allergen-specific immunotherapy (SIT) is commonly con-ducted with allergen extracts adsorbed to aluminiumhydroxide (alum). Drawbacks linked to the use of alum,such as the formation of granuloma at the site of injection,have led to suggestions of novel allergen carriers. An alter-native carrier is 2 mm carbohydrate-based particles (CBPs).In mouse, allergen-coupled CBPs have been demonstratedto skew the allergen-specific immune response towards aTh1-like activity (Gronlund et al. Immunology, 2002).We here coupled the recombinant major cat allergen Feld 1 to CBPs (CBP-Fel d 1) by cyanogen-bromide acti-vation, resulting in covalent binding. The effect of CBP-Fel d 1 on monocyte-derived dendritic cells (MDDCs)from healthy human blood donors was studied. We foundthat the majority of the CD1aþ MDDCs were capable oftaking up FITC-labelled CBP-Fel d 1, as demonstrated byflow cytometry and confocal laser scanning microscopy.Furthermore, incubation with CBP-Fel d 1 resulted in anupregulation of the costimulatory molecule CD86 on theMDDCs, which was not observed with Fel d 1 or CBPsalone. Finally, CBP-Fel d 1 induced a fivefold increasein the release of the pro-inflammatory cytokine tumournecrosis factor (TNF)-a and a fourfold increase in therelease of the chemokine interleukin-8 from MDDCs.Taken together, the effects CBPs possess make theminteresting as novel allergen carriers for SIT.


Recominant Expression andImmunological Characterization of HouseDust Mite Allergen Der P 1

H. Draborg, E. L. Roggen, N. K. Soni, S. Patkar,E. P. Friis, S. T. Lyngstrand, L. L. H. Christensen,V. Batori, S. Danielsen & S. ErnstNovozymes, Molecular Biotechnology, Bagsvaerd, Denmark.E-mail: [email protected]

The cysteine protease Der p1 from dust mite of the genusDermatophagoides pteronyssinus is a major type I allergen.About 80% of house dust mite (HDM) allergic individualsare reactive to this protease in standard assays for detectionof IgE. A curative treatment for atopic allergy is immu-notherapy (IT) with HDM extracts which are complexmixtures occasionally resulting in anaphylactic reactions.Novozymes focuses on developing a recombinant variantof Der p1 which exhibit lowered risk of IgE-mediatedallergic reactions, while maintaining its ability to triggerproper Th-cell responses. This may provide a safer

alternative for specific IT of HDM allergy. A secretedrecombinant form of pro-Der p 1 expressed by Sacchar-amyces cerevisiae was obtained by fusion of the pro-enzymeto a fungal signal peptide. The N-glycosylation site of Derp1 was mutated resulting in a deglycosylated pro-enzymewith a molecular mass of 35 kDa. Protein purificationprocedure was developed to obtain nearly pure Der p1protein followed by determination of concentration byactive-site-titration with the cysteine protease inhibitor E64.The deglycosylated recombinant pro-Der p 1 revealedimmunologic similarity to the native Der p 1 moleculewhen compared in basophile histamine release, IgE-bindingassays and T-cell proliferation assays. By in silico epitopemapping of a modelled 3-dimensional structure of Derp1, five putative IgG and IgE epitopes were predicted. Byprotein engineering, the predicted epitopes were removedone by one in Der p1 and screening for hypoallergenicvariants was performed.


Decrease in Fine T-cell Subset ratio MT2/MT1 During Steroid Reduction ofAsthmatic Patients

H. J. Hoffmann,1 L. P. Nielsen,2 G. Blumberga1 &R. Dahl1

1Department of Pulmonary Medicine, and 2Department of ClinicalPharmacology, Aarhus University Hospital, Aarhus, Denmark. E-mail:[email protected]

Combining inhaled long-acting b-2 agonist (LABA) andinhaled corticosteroid (ICS) seems to offer asthma controlat a lower dose of ICS than achieved by ICS alone. Finemapping of T-cell surface markers by flow cyto-metry offers a detailed status of the individual’sinflammatory response. The frequency of MT2(CD4þCD45RA–CD62LþCD11adim) and MT1(CD4þCD45RA–CD62L–CD11abright) cells in periph-eral blood, and their ratio, has been shown to differ pre-dictably in atopics and patients with leprosy, where MT2correlates with a Th2 phenotype and MT1 with a Th1phenotype. Stable asthmatics, requiring fluticasone pro-pionate (FP) 750–1000 mg daily or equivalent, wererandomized to receive, double-blinded, either Seretide1

[salmeterol and fluticasone propionate (SFC, n¼ 16)]50 mg/500 mg bd or FP 500 mg bd (n¼ 17). If asthmawas controlled based on lung function and symptoms atclinic visits every 6 weeks, ICS dose was tapered untilasthma exacerbated or 0 mg was reached. The frequencyand ratio of MT2 and MT1 T cells of the patients wasmonitored at 6 week intervals. As treatment tapered, thefrequency of MT2 cells decreased (P¼ 0038 from first to

Abstracts 623..................................................................................................................................................................................................


final visit), whereas that of MT1 cells increased. The ratioof MT2/MT1 decreased (P¼ 0049 from first to finalvisit). In patients receiving LABAþ ICS, the fall inMT2/MT1 ratio appeared to be more pronounced thanin patients receiving ICS alone. Thus, the MT2 phenotypemay be associated with stable asthma, whereas an immi-nent exacerbation may associate with an increase in theMT1 phenotype. LABA may allow for a greater effect ofFP on the MT ratio.


Levels, Complement Activity andPolymorphisms of Mannan-BindingLectin in Patients of Bronchial Asthmawith Allergic Rhinitis

S. Sukhija,1 V. K. Gupta,2 A. Shah,3 S. Thiel,4

P. U. Sarma1 & T. Madan1

1Institute of Genomics and Integrative Biology, Mall Road, Delhi, 2Bio-chemistry Department, Kurukshetra University, Kurukshetra, 3ClinicalResearch Centre, Vallabhbhai Patel Chest Institute, Delhi, India, and4Department of Medical Microbiology and Immunology, Aarhus University,Aarhus, Denmark. E-mail: [email protected]

Activation of complement pathways, leading to produc-tion of C3a and C5a anaphylatoxins, has been postulatedin the pathogenesis of asthma and allergic airway inflam-mation. The present study was undertaken to investigatethe role of mannan-binding lectin (MBL), an initiator ofthe lectin pathway of complement, in asthma and allergicrhinitis. MBL levels and MBL-induced complement activ-ity were determined in 19 patients of bronchial asthmawith allergic rhinitis and 20 unrelated, age-matched con-trols of Indian origin. MBL levels and activity were corre-lated with percent eosinophilia and percent predictedFEV1 values of the patients. Association of single nucleo-tide polymorphisms (SNPs) in exon 1 and intron 1 of theMBL with the disease, clinical markers, MBL levels andMBL-induced complement activity was analysed usingstandard statistical tools. Significantly higher MBL levelsand activity were observed in patients of bronchial asthmawith allergic rhinitis as compared to the controls. Weidentified five SNPs, of which two, A816G in exon 1and G1011A in intron 1 of the MBL, were novel.SNP G1011A was significantly associated with thedisease (P¼ 0.0024, OR¼ 5.8696, 95% CI:1.7316<OR< 19.8963). Individuals with ‘A’ allele atposition 1011 showed increased MBL levels, activity anddisease severity. Our results suggest that ‘A’ allele at posi-tion 1011 leading to high MBL levels and complementactivity may be contributing to the severity of bronchialasthma and allergic airway inflammation.

COMPLEMENT 1

Lysine-Dependent Binding of OspE to theC-terminus of Factor H MediatesComplement Resistance in Borreliaburgdorferi

A. Alitalo,1 T. Meri,1 H. Lankinen,2 Z.-Z. Cheng,1

S. Jokiranta,1 I. Seppala,1 P. Lahdenne,1

C. Brooks,3 P. S. Hefty,3 D. R. Akins3 & S. Meri1

1Department of Bacteriology & Immunology, Haartman Institute andHelsinki University Central Hospital, University of Helsinki, Helsinki,2Peptide and Protein Laboratory, Haartman Institute, University ofHelsinki, Helsinki, Finland, and 3Department of Microbiology andImmunology, The University of Oklahoma Health Sciences Center, Lindsay,Oklahoma City, OH, USA

Serum resistance of Borrelia burgdorferi strains belongingto the B. afzelii and B. burgdorferi sensu stricto genospeciesis dependent on binding of complement inhibitor factorH. We recently reported that factor H binding byB. burgdorferi is due to inducible expression of severalapproximately 20 kDa plasmid-encoded, surface-exposedlipoproteins related to OspE (e.g. ErpA, ErpP and P21).In addition, a second class of factor H-binding proteinsof approximately 27–35 kDa has been described. TheOspE-related lipoproteins are dramatically induced byB. burgdorferi during transmission from its tick vector intothe mammalian host. The induction of OspE-related lipo-proteins during mammalian infection may play a key a rolein the borrelial evasion of the host’s immune system. The goalof the present study was to define the factor H-bindingregions of OspE-related proteins using mutagenesis, peptidemapping and surface plasmon resonance analysis (Biacore).The combined studies revealed that the C-terminal regionsof both human and mouse factor H (SCRs 18–20) specifi-cally bind to OspE-related lipoproteins. We also foundFHR-1, whose C-terminal SCRs 3–5 are homologous toSCRs 18–20 of factor H, to bind to OspE. Peptide mappingrevealed five putative regions (designated I-V) in OspE thatcould directly interact with factor H. Deleting the C-term-inal 15 amino acid residues from region V of P21 abolishedits ability to bind factor H. At the same time, however,synthetic peptides corresponding to the C-termini of OspE,P21 and ErpP did not inhibit factor H binding to OspE.Thus, the C-terminal-binding region V appears to be neces-sary but not sufficient for factor H binding. When a morespecific mutation strategy was employed, where single aminoacid residues in peptides spanning over the factor H-bindingregions were mutated to alanines, we observed that lysines inthe factor H-binding regions of OspE were required forfactor H binding. The combined data have revealed thatkey lysine residues in OspE-related lipoproteins and ionicinteractions are crucial for factor H interactions. Further-more, binding of OspE to the C-termini of both mouse andhuman factor H suggests that Borrelia spirochetes utilize

624 Abstracts..................................................................................................................................................................................................


analogous complement resistance mechanisms in bothrodents and man. In Borrelia garinii strains, which in invitro analyses have been found to be sensitive to complementkilling, differences in the OspE sequences as well as in theexpression of factor H-binding proteins may account fortheir susceptibility to serum lysis.

COMPLEMENT 2

Role of YadA, Ail and Lipopolysaccharidein Serum Resistance of Yersiniaenterocolitica Serotype O:3

M. Biedzka-Sarek,1,2 R. Venho2 & M. Skurnik1,2,3

1Department of Bacteriology and Immunology, Haartman Institute,University of Helsinki, Helsinki, 2Department of Medical Biochemistry,University of Turku, Turku, and 3Helsinki University Central HospitalLaboratory Diagnostics, Helsinki, Finland. E-mail: [email protected]

Complement attack is one of the host strategies leading toelimination of pathogens. Yersinia enterocolitica, resistantto human serum, expresses several factors that may con-tribute to this resistance, the outer membrane proteinsYadA and Ail, and lipopolysaccharide (LPS). To studyrelative roles of these factors for the survival of Y. enter-ocolitica serotype, O:3 (Ye O:3) in nonimmune humanserum, we constructed 23 mutant strains of Ye O:3 expres-sing different combinations of YadA, Ail, LPS O-antigenand LPS outer core. Survival of bacteria was analysed innormal serum (with functional classical and alternativecomplement activation pathways) and EGTA-Mg-treatedserum (only alternative pathway functional). Kinetic killingtests revealed that the most potent single serum resistancefactor needed for long-term survival was YadA. Ail wasimportant for short-term survival and thus delayed thebacterial killing. On the contrary, the LPS O-antigen andouter core when in combination with YadA or Ail, or both,had a minor and often negative effect on serum resistance.

COMPLEMENT 3

Mannan-Binding Lectin, L-Ficolin andH-Ficolin Selectively Binds to DifferentBacteria

A. Krarup,1 U. Sørensen,1 M. Matsushita,2

J. C. Jensenius1 & S. Thiel1

1Department of Medical Microbiology and Immunology, University ofAarhus, Aarhus, Denmark, and 2Department of Applied Biochemistry andInstitute of Glycotechnology, Tokai University, Hiratsuka, Kanagawa,Japan. E-mail: [email protected]

Mannan-binding lectin (MBL), L-ficolin and H-ficolinare pattern recognition molecules of the innate immune

system. We investigated the ability of these molecules tobind to different serotypes and noncapsulated variants ofStreptococcus pneumonia and Staphylococcus aureus. Wefound that MBL binds to noncapsulated S. aureus strain(Wood) but not any of the examined S. pneumoniae ser-otypes. L-ficolin binds to some capsulated S. pneumoniaeserotypes (11A, 11D and 11F) as well as some capsulatedS. aureus serotypes (Type-1, -8, -9, -11 and -12). H-ficolindoes not bind to any of the examined S. pneumoniae andS. aureus serotypes included in this study but did bind toa strain of Aerococcus viridans. When bound to bacteria,MBL and H-ficolin initiated activation of complementfactor C4, whereas L-ficolin did not. During this study,quantitative assays for the three proteins were developedand the concentration in 97 plasma samples were deter-mined and the median values were estimated at 0.8 mg ofMBL/ml, 3.3 mg of L-ficolin/ml and 18.4 mg of H-ficolin/ml, respectively.

COMPLEMENT 4

Interferon-a mRNA in Splenic CD11b+

Marginal Zone Macrophages of C4-Deficient Mice

D. Finke,1 R. Hoerster,1 C. Brockmann,1 K. Kropf,1

H. Hennig,1 R. Zawatzky2 & S. Goerg1

1Institute for Immunology and Transfusion Medicine, University ofLuebeck, Luebeck, and 2Department of Tumorvirus-Immunology,German Cancer Research Centre, Heidelberg, Germany. E-mail:[email protected]

The absence of early complement components (C1, C4and C2 but not C3) is a predisposing factor for systemiclupus erythematosus (SLE). Recently, we demonstratedthat, in C4-deficient (C4 def.) mice, IgM-containingimmune complexes (IgM-IC) are filtered by the splenicbarrier of marginal zone macrophages (MZM), resulting inan increased immune response against antigens withinthese IgM-IC, but this could not be observed in wildtypeor C3 def. mice. We hypothesized that splenic CD11bþ

MZM play an important role in the induction of auto-immunity, and we therefore analysed their cytokine profileafter isolation with the help of magnetic antibody cellsorting. mRNA was isolated, and real-time PCR was per-formed with specific primers for murine IFN-g (IFN-g),interleukin-12 (IL-12) and IFN-a (IFN-a). We observe amoderate increase of IL-12 and IFN-g mRNA in CD11bþ

cells of C4 def. mice compared to wildtype cells. Surpris-ingly, the concentration of IFN-a mRNA is six timeshigher in C4 def. mice. Preliminary results suggest thatmRNA in CD11bþ cells of C3 def. mice is even lower thanthat in wt. Six hours following i.v. application of 20mg of a

Abstracts 625..................................................................................................................................................................................................


murine monoclonal IgM anti-dsDNA antibody, productionof IL-12, IFN-g and IFN-amRNA is increased in CD11bþ

cells of both C4 def. and wt mice. Several referencesdescribed increased levels of INF-a in patients with SLE.Dendritic cells are discussed as a major source of IFN-a.Our observation that C4-deficient, SLE-susceptible micedemonstrate an increased spontaneous IFN-a productionby splenic CD11bþ marginal zone macrophages could bean early sign and a trigger for the development of SLE. Thisis supported by the fact that the absence of C3 is not apredisposing factor for SLE and our observation that C3def. animals display low levels of IFN-a mRNA.

COMPLEMENT 5

Mannan-Binding Lectin InhibitsHumoural Responses

M. Ruseva,1 M. Gajdeva,1 K. Takahashi,2

A. Ezekowitz,2 S. Thiel1 & J. C. Jensenius1

1Department of Medical Microbiology and Immunology, University of Aarhus,Aarhus, Denmark, and 2Developmental Immunology, MassachusettsGeneral Hospital, Boston, MA, USA. E-mail: [email protected]

Chronic hepatitis B virus (HBV) infection affects about200–400 million people worldwide and represents one ofthe leading causes for liver cirrhosis and hepatocellularcarcinoma. Control over the HBV infection is achievedmainly by vaccination with Hepatitis B surface antigen(HBsAg). HBsAg contains N-linked glycosylation sideand is recognized by both MBL-A and MBL-C in a Ca-dependent manner. HbsAg–MBL complexes activate com-plement and may thus affect humoural immunity. Toinvestigate the role of MBL in humoural responses toHBsAg, we immununized mice that lack both MBL-Aand MBL-C proteins with soluble HBsAg. It has beenshown that deficiencies in other complement componentslike C1q, C4 and C3 result in decreased antibody responses.However, MBL double KO animals mounted dramaticallyincreased humoural responses. After priming, MBL doubleKOs mounted HbsAg-specific IgM responses, which werethreefold higher than WT controls. After boosting theHBsAg, total IgG was 10-fold higher in MBL KO than inWT control animals. Similar to the response to HbsAg,other glycosylated soluble antigens (e.g. invertase) inducedbetter humoural responses in MBL double KO animals,suggesting that MBL plays an important role in a negativefeedback regulation of adaptive immunity. Reconstitutionexperiments with rMBL partially rescued the KO pheno-type. We propose that the clearance of glycoprotein antigensin MBL KO is handled differently from the WT, resultingin better stimulation of humoural responses. Alternatively,glycoprotein-Ag-MBL-rich complexes inhibit B-cell respon-siveness via putative MBL receptors.

COMPLEMENT 6

Studies on the Influence of a Mutation ofMASP-2 on the Binding to MBL andFicolins

R. Sørensen, M. Gadjeva, S. Thiel & J. C. JenseniusDepartment of Medical Microbiology and Immunology, University ofAarhus, Aarhus, Denmark. E-mail: [email protected]

The complement system is an important part of the innateimmune system. The activation of complement proceedsthrough three different pathways that converge in thegeneration of C3-activating enzyme complexes. Comple-ment activation via the lectin pathway is initiated whenrecognition molecules, mannan-binding lectin (MBL) orficolin, bind to carbohydrate structures characteristic formicrobial surfaces. In the circulation, MBL and ficolins arefound in association with three structurally related MBL-associated serine proteases (MASP)-1, -2 and -3 and asmall, nonenzymatic component, MAp19. MASP-2 hasbeen shown to elicit complement activation through thesequential proteolytic cleavage of C4 and C2 upon bindingof MBL/MASP-2 complexes to microbial surfaces. Wehave recently uncovered a polymorphism in the MASP-2/MAp19 gene in a patient shown to be deficient in thelectin pathway of complement activation. The polymorph-ism results in a single amino acid substitution in theN-terminal part of the MASP-2 protein. Recombinantwildtype MASP-2 and MASP-2 containing the amino acidsubstitution in question was produced, and the ability toactivate complement was studied. The mutation had aprofound impact on MASP-2 function, resulting in thelack of complement activation through the lectin pathway.ELISA-based experiments showed that the mutation leadsto the impairment of complement activation through influ-encing the binding of MASP-2 to MBL or ficolins. Defi-ciencies in the lectin pathway of complement activation haveso far been accounted for only by lack of functional MBL.The mutation described above is the first defect describedaffecting both activation through MBL and the ficolins.

COMMENSAL S AND IMMUNITY 1

Characterization of a Large Panel ofLactic Acid Bacteria Derived from theHuman Gut for their Capacity to PolarizeDendritic Cell

H. R. Chistensen & H. FrøkiærBioCentrum-DTU, Biochemistry and Nutrition Group, Technical Universityof Denmark, kgs Lyngby, Denmark. E-mail: [email protected]

Dendritic cells (DCs) are the principal stimulators of naıveT helper (Th) cells and play a pivotal regulatory role in the

626 Abstracts..................................................................................................................................................................................................


Th1, Th2 and Treg cell balance. DCs are present in thegut mucosa and may thus be target for modulation by gutmicrobes, including ingested probiotics. Here, we testedthe hypothesis that species of lactic acid bacteria, import-ant members of the gut flora, differentially activate DC. Alarge panel of human gut-derived Lactobacillus and Bifido-bacterium spp. was screened for DC-polarizing capacity byexposing bone marrow-derived murine DC to lethallyirradiated bacteria. Cytokines in culture supernatants andDC-surface maturation markers were analysed. Substantialdifferences were found among strains in the capacity toinduce interleukin-12 (IL-12) and tumour necrosis factor(TNF)-a, while the differences for IL-10 and IL-6 wereless pronounced. Bifidobacteria tended to be weak IL-12and TNF-a inducers, while both strong and weak IL-12inducers were found among the strains of Lactobacillus.Remarkably, strains weak in IL-12 induction inhibitedIL-12 and TNF-a production induced by an otherwisestrong cytokine-inducing strain of Lactobacillus casei,while IL-10 production remained unaltered. Selectedstrains were tested for induction of DC maturation mar-kers. Those lactobacilli with greatest capacity to induce IL-12 were most effective in upregulating surface MHC classII and CD86. Moreover, L. casei-induced upregulation ofCD86 was reduced in the presence of a weak IL-12-inducing L. reuteri. In conclusion, human Lactobacillusand Bifidobacterium spp. polarize differentially DC matur-ation. Thus, the potential exists for Th1/Th2/Treg-drivingcapacities of the gut DC to be modulated according tocomposition of gut flora including ingested probiotics.

COMMENSALS AND IMMUNITY 2

Human Isolates of Lactic Acid BacteriaDifferentially Affect Maturation andCytokine Production by Human DendriticCells

L. Hjerrild, H. R. Christensen & H. FrøkiærBiochemistry and Nutrition Group, BioCentrum-DTU, Technical Universityof Denmark, Lyngby, Denmark. E-mail: [email protected]

The intestinal micro flora is indispensable in developingand maintaining homeostasis of the gut-associatedimmune system. Evidence indicates that lactic acid bacteria(LAB), e.g. lactobacilli and bifidobacteria, have beneficialeffects on the host. Established health effects includeincreased gut maturation, antagonisms towards pathogensand immune modulation. The objective of this study isto evaluate the immunomodulating properties of a rangeof LAB of human origin. As dendritic cells (DCs) playa pivotal role in the balance between tolerance andimmunity to commensal microorganisms, in vitro-generated

immature DCs serve as a suitable model for studying theimmunomodulating effects of lab. Human immature DCswere generated in vitro from monocytes and exposed tolethally UV-irradiated LAB. The effect of various species ofLAB on DCs in direct contact was evaluated. Furthermore,the maturation pattern of DCs separated from the bacteriaby an epithelial cell layer (CaCo-2 cells), which shouldmimic the intestinal environment, was studied. Cytokinesecretion (IL-12, IL-10 and TNF-a) and upregulation ofmaturation surface markers on DCs (CD83 and CD86)was measured. Different LAB induced diverse cytokineresponses. Some strains were strong IL-12 and TNF-ainducers and others weak. All strains induced IL-10. Differ-ent LAB also differentially modulated expression of CD83and CD86 on DCs. Although some variation in the responseto LAB of DCs generated from different blood donors wasobserved, general differences in the effect of the various LABwas revealed. Experiments with the DC CaCo-2 coculturesystem are ongoing. Different species of LAB differentiallyaffect DC maturation; this suggets that the gut flora plays apivotal role in polarization of the immune response.

COMMENSALS AND IMMUNITY 3

Lactobacilli Modulate Proliferation andCytokine Production of Human PeripheralBlood Natural Killer Cells In Vitro

L. N. Fink, H. R. Christensen & H. FrøkiærBiochemistry and Nutrition Group, BioCentrum-DTU, Technical Universityof Denmark, Lyngby, Denmark. E-mail: [email protected]

Natural killer (NK) cells are cells of the nonspecificimmune system lysing altered self-cells. A noncytolyticsubset of NK cells may serve a regulatory role by secretingcytokines. Bacteria translocating across the gastrointestinalmucosa are presumed to gain access to NK cells, as con-sumption of certain lactic acid bacteria has been shown toincrease in vivo NK cytotoxicity. Here, we investigatedhow human gut flora-derived lactobacilli affect NK cellsin vitro, by measuring proliferation and IFN-g productionof human NK cells upon bacterial stimulation.CD3–CD56þNK cells were isolated from buffy coats bynegative isolation using non-NK lineage-specific anti-bodies and magnetic beads. NK cells were incubated with10mg/ml UV-inactivated bacteria or 10mg/ml phytohemag-glutinin (PHA) for 4 days. Proliferation was assessedby incorporation of radioactive thymidine into NK-cellDNA. The IFN-g concentration was measured by ELISA.Incubation of NK cells with a Lactobacillus acidophilus strainincreased the proliferation of the NK cells and inducedIFN-g production, both to levels comparable to PHA

Abstracts 627..................................................................................................................................................................................................


stimulation. The proliferative response was further enhancedwith autologous monocytes present, probably becausecytokines, secreted by monocytes having engulfed bacteria,stimulated the NK cells. In contrast, a Lactobacillus paracaseistrain caused the NK cells to proliferate only in the presenceof monocytes. These results demonstrate that various strainsof lactobacilli have the capacity to activate NK cells in vitro,in a monocyte-dependent or -independent way. Hence, theencounter of NK cells with lactic acid bacteria will affectNK-cell activation. Such activation of NK cells may poten-tially skew an on-going or subsequent immune responsetowards a Th1 response.


Idiotypic and Anti-Idiotypic AntibodiesProduced in Immune Response to BacteriaLactobacillus acidophyllus

S. Zivancevic-Simonovic,1 A. Inic-Kanada,2

M. Petricevic,2 L. J. Mijatovic1 & L. J. Dimitrijevic2

1School of Medicine, University of Kragujevac, Kragujevac and 2Instituteof Immunology and Virology, Torlak, Belgrade, Serbia and Montenegro.E-mail: [email protected]

Lactobacilli are nonpathogenic gram-positive inhabitantsof the normal human intestine known for their health-promoting effects. In our earlier work, it is shown thathuman monoclonal antibody isolated from sera of apatient with Waldenstrom macroglobulinaemia possessinnate antibody characteristics and binds to lactic acidbacteria. According to the immune network model, immu-nization with this bacteria could induce the perturbationsin immune system that might result in production of anti-Lactobacillus antibodies, human monoclonal antibody like(Ab1) and anti-idiotypic antibody (Ab2). In this study,BALB/c mice were immunized with two doses of bacteriaLactobacillus acidophilus in complete and incompleteFreund’s adjuvant and phosphate-buffered saline (PBS),respectively. Seven days after the last immunization, serafrom immunized mice were collected and the presence ofLactobacillus-specific Ab1 and Ab2 were determined byELISAs. In the sera of immunized mice, antibodies specificto bacteria Lactobacillus acidophilus were shown. The con-centration of Lactobacillus-specific antibodies was higherin the sera of hyperimmunized mice (mice immunizedwith 1mg of IgM DJ) than in sera of mice immunizedwith 100 times lower doses of immunogen (0.01mgper doses). Moreover, Ab1 and Ab2 antibodies weredetected in the sera of Lactobacillus-hyperimmunizedmice. In this study, we have shown the idiotypic networkinteractions in mice immunized with bacteria Lactobacillusacidophilus.


Pattern of Cytokine Responses to Gram-Positive and Gram-Negative CommensalBacteria is Profoundly Changed whenMonocytes Differentiate into DendriticCells

H. Karlsson,1 P. Larsson,1 A. E. Wold2 & A. Rudin1

1Department of Rheumatology and Inflammation, and 2Department ofClinical Bacteriology, Goteborg University, Goteborg, Sweden. E-mail:[email protected]

The normal gastrointestinal flora is crucial for the matur-ation of the acquired immunity via effects on antigen-presenting cells (APCs). Here, we have investigated howtwo types of APCs, monocytes and dendritic cells (DCs),react to different bacterial strains typical of the commensalintestinal flora. Purified monocytes and monocyte-derivedDCs were stimulated with UV-inactivated gram-positive(Lactobacillus plantarum and Bifidobacterium adolescentis)and gram-negative (Escherichia coli and Veillonella parvula)bacterial strains. Monocytes produced higher levels of IL-12p70 and TNF, as detected by ELISA, in response to L.plantarum than to E. coli and V. parvula. In contrast, DCssecreted high amounts of IL-12p70, TNF, IL-6 and IL-10in response to E. coli and V. parvula but were practicallyunresponsive to L. plantarum and B. adolescentis. The lackof response to the gram-positive strains correlated with alower surface expression of Toll-like reseptor 2 (TLR2) onDCs compared to monocytes. The surface expression ofTLR4 on DCs was undetectable when analysed by flowcytometry, but blocking this receptor decreased the TNFproduction in response to V. parvula, indicating that lowTLR4 expression on DCs is sufficient to mount an inflam-matory response to gram-negative bacteria. IFN-gincreased the expression of TLR4 on DCs and also poten-tiated the cytokine response to gram-negative bacteria.Our results indicate that, when monocytes differentiateinto DCs, their ability to respond to different commensalbacteria dramatically changes, thereby becoming unrespon-sive to probiotic gram-positive bacteria. These results mayhave important implications for the capacity of differentgroups of commensal bacteria to regulate mucosal andsystemic immunity.


Probiotic Bacteria Induce RegulatoryCytokine Production via Dendritic Cells

C. L. Hvas,1 J. Kelsen,1 J. Agnholt,1 P. Hollsberg2 &J. F. Dahlerup1

628 Abstracts..................................................................................................................................................................................................


1Department of Medicine V, Aarhus University Hospital, Arhus Sygehus,and 2Department of Medical Microbiology and Immunology, University ofAarhus, Aarhus, Denmark. E-mail: [email protected]

Probiotic bacteria, e.g. Lactobacillus spp., may improvediseases such as chronic inflammatory bowel disease. Weexamined cytokine production and phenotypic changeafter in vitro stimulation of T cells from healthy volunteersusing different probiotic strains.Methods: T cells were cultured from colonic biopsiesfrom eight healthy volunteers (Agnholt and Kaltoft, ExpClin Immunogenet 2001;18:213–25), and dendritic cellswere matured from their peripheral blood mononuclearcells. T-cell cultures were stimulated with autologous bac-terial sonicate or strains of Lactobacillus spp., with andwithout the addition of dendritic cells. Cytokine levels(TNF-a, IFN-g, IL-10 and GM-CSF) and phenotype(CD3, CD4, CD25 and CD69) were measured on day 4.Results: Lactobacillus spp. induced higher productions ofTNF-a and IL-10 than did autologous bacteria. In pre-sence of dendritic cells, the production of all cytokinesincreased. However, the increases of IFN-g and TNF-awere more pronounced in wells with autologous bacteriathan in wells with Lactobacillus spp. The addition ofdendritic cells upregulated CD25 expression withoutsimultaneous upregulation of CD69. The upregulationwas pronounced after stimulation with Lactobacillusrhamnosus GG compared with autologous bacteria andother lactobacilli.Discussion: In presence of dendritic cells, autologous bac-teria induced inflammatory cytokines, while probioticsmainly induced regulatory cytokines. Lactobacillus rham-nosus GG induced a regulatory phenotype (cd25þ), in partmediated by dendritic cells. Future studies will addresswhether this shift to a CD25þ phenotype represents adifferentiation into competent regulatory T cells. In aclinical context, such cells might be used for treatment ofinflammatory diseases.

IMMUNOTECHNOLOGY 1

Proteome Analysis Based on HumanRecombinant Antibody Microarrays

J. Ingvarsson, M. Lindstedt, C. A. K. Borrebaeck &C. WingrenDepartment of Immunotechnology, Lund University, Lund, Sweden. E-mail:[email protected]

Protein microarrays will play a key role in the postgenomicera and offer a unique possibility to perform high-throughput global proteome analysis. A chip can be printedwith thousands of protein probes (e.g. antibodies), thebiological sample added (e.g. a proteome) and any bindingdetected. We aim to develop protein microarrays based on

human recombinant scFv antibody fragments for globalproteome analysis. The concept of comparing proteomicmaps of healthy versus diseased samples will allow disease-specific proteins to be detected. In fact, antibody microarrayswill allow us to perform comparative proteome analysis onany sample format in a species-independent manner, as longas a proteome can be isolated. However, the complexity ofproteomes, containing several thousands of different pro-teins, is a problem. Here, we have designed antibody micro-arrays targeting the water-soluble fraction of a proteome. Tothis end, an anticytokine antibody array was developed andhuman dendritic cells (�activation) was used as model sys-tem. The results showed that our antibody microarrays couldbe used to examine the cytokine profile in complex samples.Furthermore, we have taken the first steps towards compar-ing our results with those of other technologies on both theprotein and gene level. Due to the complexity of the modelproteome, we also examined the possibility to prefractionatethe proteome in a simple one-step procedure (based on size)prior to the labelling step. In more detail, the sample pro-teome was fractionated into two fractions using membranedevices with different molecular weight cut-offs. The resultsshowed that the fractionation considerably enhanced theassay sensitivity allowing cytokines in the pg/ml range tobe readily detectable.

IMMUNOTECHNOLOGY 2

The Immunomodulatory Effect of HeatShock Protein 70: Immunization with aDNA Construct Based on the MalarialAntigen Fused with a Fragment of HSP 70Primes for a Th-1 Type of Response

Q. K. Rahman,1 M. Wikman,2 N.-M. Vasconcelos,1

K. Berzins,1 S. Stahl2 & C. Fernandez1

1Department of Immunology, Stockholm University, Stockholm, Sweden,and 2Department of Biotechnology, Kungl Tekniska Hogskolan, Stockholm,Sweden. E-mail: [email protected]

Finding an appropriate adjuvant for human vaccination iscrucial. Heat shock proteins (HSPs) act as adjuvants whencoadministered with peptide antigens or given as fusionproteins. However, there is a potential risk of autoimmun-ity when using the complete molecules, because HSPs areevolutionary conserved. To overcome this, we first evalu-ated the adjuvant effect against two different antigens of aless-conserved fraction of Plasmodium falciparum HSP70(Pf70C) and compared it to the whole HSP70 moleculefrom Trypanosoma cruzi (TcHSP70). We found thatPf70C exhibited similar adjuvant properties as the wholemolecule. We later evaluated the adjuvant potential ofPf70C against the malarial antigen EB200 in a chimericDNA construct. No appreciable levels of EB200-specific

Abstracts 629..................................................................................................................................................................................................


antibodies were detected in mice immunized only with theDNA constructs. However, DNA primed the immunesystem, because subsequent challenge with the correspond-ing recombinant fusion proteins elicited a strong Th-1antibody response. In contrast, no priming effect wasobserved for ex vivo IFN-g production but stimulationwith the HSP-chimeric fusion protein induced a strongersecretion of IFN-g in vitro than other proteins used. Theseresults indicate that the use of HSPs is promising in thedesign of new vaccines.

IMMUNOTECHNOLOGY 3

High-Throughput Proteomics onAntibody-based Microarrays: theImportance of Probe and Surface Design

C. Steinhauer, C. Wingren & C. A. K. BorrebaeckDepartment of Immunotechnology, Lund University, Lund, Sweden. E-mail:[email protected]

In analogy to DNA microarrays, protein microarrays offera new distinct possibility to perform sensitive high-throughput global proteome analysis. However, the devel-opment of the protein microarray technology will placehigh demands upon the design of both probes and solidsupports. The analysis of thousands of heterogeneous pro-teins on a single microarray requires the use of uniformprobes, such as antibodies, directly designed for proteinmicroarray applications. We have recently generated ahuman recombinant single-chain Fv antibody library,genetically constructed around one framework, thenCoDeR-library, containing 2� 1010 clones. Single frame-work antibody fragments (sinFabs) selected from thislibrary were successfully applied as probes for microarraysproviding sensitive detection in the 600 attomol (massspectrometry) and the 300 zeptomole range (fluorescence).However, the choice of framework is critical. We haveshown that the selected nCoDeR framework displayedexcellent functional on-chip stability and arrayed dehy-drated probes retained their activity for several months.Furthermore, we have addressed the issues of biocompati-bility of the solid support and immobilization strategiesfor our microarray setup. An in-house-designed substrate,macroporous silicon coated with nitrocellulose (MAP3-NC7), displayed properties equal to, or better than, thoseof five commercially available supports used as referencesurfaces. We have also evaluated different coupling strat-egies, such as adsorption, covalent coupling, diffusionand affinity coupling. Using a novel affinity tag, thedouble-(his)6-tag, we increased the binding efficiency ofsinFab-molecules to Ni2þ-coated solid supports, therebyallowing nonpurified probes to be directly applied.

IMMUNOTECHNOLOGY 4

An Assay for Mannan-Binding Lectin-Associated Serine Protease 3, MASP-3

S. H. Holmvad, M. Dahl, J. C. Jensenius & S. ThielDepartment of Medical Microbiology and Immunology, University ofAarhus, Denmark. E-mail: [email protected]

The mannan-binding lectin (MBL) pathway is part of theinnate immune system providing a first line of defenceagainst infections. MBL and ficolins circulate in complexeswith MBL-associated serine proteases (MASP-1, -2 and-3). After recognition of a microorganism by MBL, activa-tion of the complement system occurs. MASP-1 andMASP-3 share five domains (making up the so-calledA-chain), whereas they have unique protease domains(B-chains). Before the identification of MASP-3, an assayfor MASP was presented, based on antibodies against theA-chain of MASP-1. With the new knowledge of the threeMASPs, and the sharing of domains by MASP-1 andMASP-3, assays specific for the protease domains have tobe constructed, if one wishes to measure the proteinsindividually. We present an assay for quantifying totalMASP-3 in plasma and serum samples. The assay is asandwich-type assay using as catching antibody a mono-clonal antibody against the common A-chain of MASP-1/3 and a developing secondary antibody against theC-terminal part of the protease domain of MASP-3. Wehave used this assay for estimating the normal concentra-tion of the protein as well as the concentration in patientsand also for characterizing by gel permeation chromato-graphy the MASP-3 protein in serum.

IMMUNOTECHNOLOGY 5

The Human-Inducible CostimulatorLigand is Polymorphic

K. Jacobsen, L. Ohm-Laursen & T. BaringtonDepartment of Clinical Immunology, Odense University Hospital, Odense,Denmark. E-mail: [email protected]

Inducible costimulator ligand (ICOSL) is a costimulatorymolecule related to B7.1 (CD80) and B7.2 (CD86).B cells, monocytes, dendritic cells and endothelial cellsexpress ICOSL. Inducible costimulator (ICOS) interactswith ICOSL, and this interaction leads to signals involvedin isotype switching and the development of immuno-logical memory. Hitherto, no polymorphisms of this genehave been described. The aim of this study was to revealvariation of the ICOSL gene in normal individuals. Alleight exons, except exon 1, were sequenced with flankingintrons in 10 healthy blood donors. Eight single nucleotidepolymorphisms (SNPs) and two length polymorphismswere found. One of the SNPs was found in the coding

630 Abstracts..................................................................................................................................................................................................


regions of the gene. The base involved was located in exon3 and caused a conservative amino acid change from valine(GTT) to isoleucine (ATT). Three individuals were het-erozygous G/A for the exon polymorphism, while theremaining seven individuals were homozygous for thewildtype G/G. Exon 3 encodes the immunoglobulin vari-able (IgV)-like domain of the molecule which is situatedoutside the cell. This means that the amino acid could becritical for the stability of the molecule or could constitutepart of the binding site for ICOS. The results form thebasis for further experiments to find possible associationsof the alleles to diseases caused by immune dysregulation.Especially, the exon 3 variant is interesting and could playa role for the development of immunological diseases.Besides, it would be interesting to see whether bothexon 3 alleles are expressed or only the wildtype allele isfunctional.

IMMUNOTECHNOLOGY 6

Monitoring Patients Treated with Type1Interferons: Potential Reporter Genes inPatient Leucocytes

S. U. S. Sønder, C. J. Hedegaard & K. BendtzenInstitute for Inflammation Research, Rigshospitalet University Hospital,Copenhagen, Denmark. E-mail: [email protected]

Interferon-a/b (IFN-a/b) is increasingly used as antiviraland immunomodulatory therapies. Unfortunately, bio-availability varies with IFN species and mode of adminis-tration, and all IFN species are potentially immunogenic.Assays for antiviral activity (IFN) and antiviral neutraliza-tion (antibodies, NAb) have been used for some time tomonitor patients on IFN biologicals. These assays requirelaborious titrations making them unsuitable for large-scaleclinical use. Myxovirus A (MxA) is a resistance GTP-binding protein that is specifically induced by treatmentwith type 1 IFNs. For example, IFN-b-induced MxA inblood leucocytes has been used as a biomarker in IFN-b-treated patients with multiple sclerosis. However, thedegree of specificity of MxA in this regard is unclear, andmeasurements of MxA protein and/or mRNA are not yetsuitable for routine clinical use. In an attempt to find newand better reporter genes (and, hopefully, genes and geneproducts with proven specificity for IFN-a and -b), micro-array screenings with U133A GeneChips (Affymetrix)were carried using human blood leucocytes and thehuman lung carcinoma cell line A549. We studied thesimultaneous expression of 22,000 transcripts before andafter exposure to human recombinant IFN-a and IFN-band other antiviral and immunomodulatory cytokines.The results will be presented at the conference.

IMMUNOTECHNOLOGY 7

Monitoring Patients Treated with Type 1Interferons: Antiviral versus MxAInduction Assays

S. U. S. Sønder, C. J. Hedegaard & K. BendtzenInstitute for Inflammation Research, Rigshospitalet University Hospital,Copenhagen, Denmark. E-mail: [email protected]

Interferon-a/b (IFN-a/b) is increasingly used as antiviraland immunomodulatory therapies. Unfortunately, bio-availability varies with IFN species and mode of adminis-tration, and all IFN species are potentially immunogenic.Assays for antiviral activity (IFN) and antiviral neutraliza-tion (antibodies, NAb) have been used for some time tomonitor patients on IFN biologicals. These assays requirelaborious titrations making them unsuitable for large-scaleclinical use. Our laboratories have therefore modified theantiviral assays for IFN bioactivity and Nab, so that theyare suitable for large-scale screening in specialized labora-tories. The read-out is survival of a subcloned A549 cellline in the presence of an otherwise lethal amount of virus.Thus, survival increases in the presence of type 1 IFN anddecreases in the presence of NAb against the IFN added tothe cells. MxA is induced by type 1 IFN and can be usedfor measuring the Nab activity. In another assay, the MxAlevel in the A549 cell line is measured. In an attempt tofind a new and better reporter gene for type 1 IFN thanMxA and genes specific for either IFN-a or -b, a microarray screen was carried using the U133A chip from Affy-metrix. The expression of 22,000 genes can be studiedsimultaneous with this technology. The results will bepresented at the conference.

IMMUNOTECHNOLOGY 8

A Database Solution for LaboratoryInformation Management

M. S. Petersen, C. C. Fleischer, R. Agger &M. HoklandDepartment of Medical Microbiology and Immunology, Aarhus University,Aarhus, Denamark. E-mail: [email protected]

In our laboratory, we have developed a database system,which we believe is of immediate interest to the generalscientific community. The database represents a computer-based replacement for the laboratory notebooks used inthe majority of research laboratories worldwide. In addition,the database provides an effective tool for organizing andmanaging laboratory information at all levels, spanningfrom managing and revising standard operating proceduresand producing documentation of research activities to keep-ing track of data and conclusions. Using the commercially

Abstracts 631..................................................................................................................................................................................................


available database toolkit software FILEMAKER PRO, we havedeveloped a relational database solution for managementof laboratory information. The system consists of ahierarchy of five interrelated databases, each pertainingto a separate type of information, namely, overall projectinformation, information relating to individual experimentsetups, documentation of daily research activity, generateddata and descriptions of standard operating procedures.Like other databases, each individual database consists ofa number of records, each comprised of a set of fields inwhich information is entered. In each record, a certainfield is reserved to specify the relation of the record to arecord in another database at a higher level. Thus, thedatabase is essentially five databases linked by a hierarchyof one-to-many relations, organizing information in afolder-like structure. Importantly, the database systemallows multiple users to access and edit records simulta-neously, and the data entered in one database immediatelybecomes accessible through the other databases. The limi-tations of laboratory notebooks are apparent when look-ing for information, which is dispersed throughout one ormore notebooks, or possibly on loose sheets of paper orprintouts ‘somewhere’. The often complicated process ofgathering laboratory data or results when writing grantapplications or research papers is made considerably easierwith the database system. Thus, the database solutionpresented should be broadly attractive to researchers, irre-spective of their scientific discipline.

IMMUNOTECHNOLOGY 9

SARS CTL Vaccine Candidates — HLASupertype, Genome-Wide Scanning andBiochemical Validation

C. Sylvester-Hvid,1 M. Nielsen,2 K. Lamberth,1

G. Røder,1 S. Justesen,1 C. Lundegaard,2

P. Worning,2 H. Thomadsen,1 O. Lund,2 S. Brunak2

& S. Buus2

1Division of Experimental Immunology, Institute of Medical Microbiologyand Immunology, University of Copenhagen, Copenhagen and 2Center forBiological Sequence Analysis (CBS), Technical University of Denmark,Lyngby, Denmark. E-mail: [email protected]

An effective SARS vaccine is likely to include componentsthat can induce specific cytotoxic T-cell (CTL) responses.The specificities of such responses are governed by HLA-restricted presentation of SARS-derived peptide epitopes.Exact knowledge of how the immune system handlesprotein antigens would allow for the identification ofsuch linear sequences directly from genomic/proteomicsequence information. The latter was recently establishedwhen a causative coronavirus (SARS CoV) was isolatedand full-length sequenced. Here, we have combined

advanced bioinformatics and high-throughput immun-ology to perform an HLA supertype, genome-wide scanfor SARS-specific cytotoxic T cell epitopes. The scanincludes all nine human HLA supertypes in total covering>99% of all major human populations. For each HLAsupertype, we have selected the 15 top candidates for testin biochemical-binding assays. At this time (approximately6 months after the genome was established), we have testedthe majority of the HLA supertypes and identified almost100 potential vaccine candidates. These should be furthervalidated in SARS survivors and used for vaccine formula-tion. We suggest that immunobioinformatics may becomea fast and valuable tool in rational vaccine design.

IMMUNOTECHNOLOGY 10

Production of Functionally ActiveRecombinant HLA MoleculesRepresenting the Supertypes B62 andB58 and the Generation of CorrespondingPeptide-Binding Assays and Tetramers

G. Røder, K. Lamberth & S. BuusDepartment of Medical Microbiology and Immunology, Panum Institute,University of Copenhagen, Copenhagen, Denmark. E-mail: [email protected]

Rationale: Major histocompatibility complex class I(MHC I) molecules monitor the protein content of thecell by binding small derived peptides and presenting themto cytotoxic CD8þ T cells. The goal of the human MHCproject is to predict the binding strength of any givenpeptide/MHC complex. This prediction allows the designof peptide-based vaccines. The prediction requires repre-sentative binding data from MHC alleles from all the nineHLA supertypes. Here, we describe the genetic construc-tion, protein production and purification as well as theestablishment-binding assays for two recombinant MHCsupertype alleles, HLA-B*1501 and HLA-B*5801.Methods: Using the Quikchange Multisite DirectedMutagenesis Kit (Stratagene), codon-optimized genesencoding HLA-B*1501 and HLA-B*5801 are created.The two MHC I molecules are fermented and purifiedby ion exchange chromatography, hydrophobic interactionchromatography and size exclusion chromatography. Thebinding (KD) of natural T-cell epitopes, as well as pre-dicted peptide ligands, is described by radioactive immuno-assays (RIAs) and enzyme-linked immunosorbent assays(ELISAs). The MHC molecules are biotinylated duringexpression.Results: The expression of MHC I resulted in multipledisulfide bond isomers, which are separated by hydropho-bic interaction chromatography and used in subsequentbinding studies resulting in the determination of KD forvarious peptide ligands ranging from strong binders

632 Abstracts..................................................................................................................................................................................................


(KD< 50 nM) to low binders (KD> 5 mM). Tetrameriza-tion is visualized by SDS-PAGE.Conclusion: An effective method for the production ofhighly pure MHC I molecules has been applied to HLA-B*1501 and HLA-B*5801, and RIA and ELISA bindingassays for those alleles have been established, showing thebinding of various peptide ligands to the MHC I molecules.

IMMUNOTECHNOLOGY 11

‘Query-by Committee’ — An EfficientMethod to Select Information-Rich Datafor the Development of Peptide—HLA-Binding Predictors

K. Lamberth,1 M. Nielsen,2 C. Lundegaard,2

P. Worning,2 S. L. Laurmøller,1 O. Lund,2

S. Brunak2 & S. Buus1

1Department of Experimental Immunology, Institute of Medical Micro-biology and Immunology, University of Copenhagen, Copenhagen,Denmark, and 2Center for Biological Sequence Analysis, BioCentrum-DTU,Technical University of Denmark, Lyngby, Denmark. E-mail:[email protected]

Rationale: We have previously demonstrated that bioin-formatics tools such as artificial neural networks (ANNs)are capable of performing pathogen-, genome- and HLA-wide predictions of peptide–HLA interactions. These toolsmay therefore enable a fast and rational approach to epi-tope identification and thereby assist in the developmentof vaccines and immunotherapy. A crucial step in the gener-ation of such bioinformatics tools is the selection of datarepresenting the event in question (in casu peptide–HLAinteraction). This is particularly important when it isdifficult and expensive to obtain data. Herein, we demon-strate the importance in selecting information-rich dataand we develop a computational method, query-by-committee, which can perform a global identification ofsuch information-rich data in an unbiased and automatedmanner. Furthermore, we demonstrate how this methodcan be applied to an efficient iterative developmentstrategy for these bioinformatics tools.Methods: A large panel of binding affinities of peptidesbinding to HLA A*0204 was measured by a radioimmuno-assay (RIA). This data was used to develop multiple firstgeneration ANNs, which formed a virtual committee. Thiscommittee was used to screen (or ‘queried’) for peptides,where the ANNs agreed (‘low-QBC’), or disagreed (‘high-QBC’), on their HLA-binding potential. Seventeen low-QBC peptides and 17 high-QBC peptides were synthesizedand tested. The high- or low-QBC data were added to theoriginal data, and new high- or low-QBC second generationANNs were developed, respectively. This procedure wasrepeated 40 times.

Results: The high-QBC-enriched ANN performed signif-icantly better than the low-QBC-enriched ANN in 37 ofthe 40 tests.Conclusion: These results demonstrate that high-QBC-enriched networks perform better than low-QBC-enrichednetworks in selecting informative data for developingpeptide–MHC-binding predictors. This improvement inselecting data is not due to differences in network trainingperformance but due to the difference in informationcontent in the high-QBC experiment and in the low-QBC experiment. Finally, it should be noted that thisstrategy could be used in many contexts where generationof data is difficult and costly.

TUMOUR IMMUNOLOGY 1

Interleukin-18 Inhibition of OralCarcinoma Cell Proliferation

A. Nilkaeo & S. BhuvanathDepartment of Microbiology, Faculty of Science, Prince of Songkla Uni-versity, Songkla, Thailand. E-mail: [email protected]

Interleukin-18 (IL-18), a pro-inflammatory cytokine thatis produced by both lymphoid and nonlymphoid cells, hasa critical role in modulation of innate and adaptive immun-ity. Its primary function in stimulation of IFN-g produc-tion and stimulation of NK-cell-cytotoxic activities makesthis cytokine a candidate for cancer immunotherapy. Inoral cavity, this cytokine is produced by oral epithelia andcarcinoma cells and is related to tumour regression in nudemice bearing salivary adenocarcinoma. However, directeffects of this cytokine on oral cancer cells have not beenelucidated. In this project, we investigated IL-18 effect onan oral carcinoma (KB) cell line. With RT-PCR techni-que, KB-cell line was found to express IL-18 receptors(IL-18Ra and IL-18Rb), indicating that this oral carci-noma line is a target for IL-18 study. We showed thatrecombinant human IL-18 inhibited KB-cell proliferationby 17% at concentration of 100 ng/ml (P< 0.05), whereasLDH release by these cells in treatment group and controlgroups was comparable, indicating that IL-18 suppressionof cell proliferation was not mediated by the induction ofcell death. To further address this hypothesis, we foundthat IL-18 treatment did not induce apoptotic cell death,as studied by DNA laddering and TUNEL assays. Inaddition, expression pattern of cell death-controllinggenes (bcl-2 and bax) was not altered by this cytokine.Findings in these studies indicated that suppression ofKB-cell proliferation may be attributed to control of cellcycle, growth arrest or induction of cell differentiation.The data presented in this project could provide an insightof how cancer cell directly responds to IL-18, as this cyto-kine is an important regulator of anticancer mechanisms.

Abstracts 633..................................................................................................................................................................................................


TUMOUR IMMUNOLOGY 2

BCL-6 Prevents Mammary EpithelialApoptosis, Promotes Cell Survival and isExpressed in Breast Carcinoma

F. Q. B. AlenziDepartment of Medical Laboratory Sciences, College of Applied MedicalSciences, King Faisal University, Dammam, Saudi Arabia. E-mail:[email protected]

Background: Proliferation, differentiation and apoptosisare essential processes in the normal functions of themammary epithelium. The hypothesis examined in thisstudy is that the transcription factor BCL-6 is criticallyimportant not only for regulating B-cell growth and devel-opment but also for mammary epithelial apoptosis.Methodology: Twenty breast cancer cases and 31 healthycontrols were used to investigate whether BCL-6 protein ininvolved in breast cancer (grade III). Full length BCL-6cDNA was retrovirally transduced into EpH-4 cell line. Wethen used flow cytometry of BrdUrd-stained cells to investi-gate the cell-cycle duration of the control and transduced celllines. TUNEL was used as a marker of apoptosis to find outdifferences in the frequencies of apoptotic cells in the controland transduced cell lines. Finally, immunohistochemistrystaining was performed to detect BCL-6 in breast cancer (III).Results: Restoration of BCL-6 into EpH-4 cells not onlyinhibits apoptosis but also prolongs the cell cycle and resultsin increased cell size and protein content. The results alsoindicated that the cell-cycle time of BCL-6-transducedEpH-4 cells is prolonged by about 3 h, presumably as aresult of the action of BCL-6 at the BCL-6 at the G1/Stransition. We found differences in the frequencies of viableand apoptotic cells in cultures of the parent EpH-4 cells,control-transduced EpH-4 cells and BCL-6-transducedEpH-4 cells. Consistently, we demonstrated that BCL-6 isexpressed in 90% of high grade of breast carcinoma, whichis considered as the most aggressive of tumours.Conclusion: Together, these results suggest that BCL-6 islikely to be involved in mammary gland development andcarcinogenesis.

TUMOUR IMMUNOLOGY 3

Inflammatory Cytokine Modulation ofCancer Cell Proliferation

S. Bhuvanath & A. NilkaeoDepartment of Microbiology, Faculty of Science, Prince of SongklaUniversity, Songkla, Thailand. E-mail: [email protected]

Inflammatory cytokines have a critical role in modulationof both innate and adaptive immunity in response to

foreign antigen. They also play an important role in anti-cancer immunity. For example, they canpromote cell-mediatedimmunity against cancer cells. With their immuno-stimulatory effects, these cytokines are being tested for cancertreatment in the form of DNA vaccine or adjuvant ortherapeutic cytokines. Direct effect of these cytokines oncancer cell, however, is still unclear. In this project, weinvestigated whether IL-1( and IL-18 can modulate cancercell proliferation. We employed a simple nonradioactiveproliferation (MTT) assay and detection of lactate dehydro-genase (LDH) to test the effect of these recombinant humancytokines on various cancer cell lines, including breast cancercell line (MCF-7), oral carcinoma cell line (KB), coloncancer cell line (Caco-2) and choriocarcinoma cell line(Jar). Cytokines used in this study had both inhibitory andstimulatory effect on cell proliferation. Findings in this pro-ject could provide an insight of cancer cell response to thesecytokines and this could lead to a consideration on usingcytokine as immunotherapy for cancer treatment.

TUMOUR IMMUNOLOGY 4

Capacity of AE to Modulate Nitric OxideProduction Depended on IntercellularContact

S. Mijatovic,1 D. Maksimovic-Ivanic,1

D. J. Miljkovic,1 L. J. Harhaji1 & V. Trajkovic2

1Institute for Biological Research ‘Sinisa Stankovic’, and 2Institute ofMicrobiology and Immunology, School of Medicine, University of Belgrade,Belgrade, Serbia. E-mail: [email protected]

Aloe emodin (AE) is a naturally occurring compound withwide spectrum of biological properties, including anti-microbial, vasorelaxant, immunosuppressive and antican-cer actions. This anthraquinone induces apoptosis inseveral tumour cell lines with special affinity to tumoursof neuroectodermal origin. High amounts of nitric oxide(NO) released by activated macrophages induce tumourcell death. Therefore, we explored the capacity of AE tomodulate NO-mediated antitumour response in vitro.Interestingly, while AE markedly suppressed NO releasefrom macrophages alone, it significantly potentiated NOproduction in cocultures of macrophages and C6 cells,after 48 h of cultivation. Accordingly, the viability of C6cells cocultivated with macrophages was reduced in thepresence of AE. Moreover, the observed AE-imposedpotentiation of NO production in macrophages was clo-sely related to macrophage culture cell density. Accordingto these data, we proposed that NO modulator capacity ofAE strongly depended on intercellular contact, indicatingthat macrophage antitumour response was not compro-mised but even potentiated by AE.

634 Abstracts..................................................................................................................................................................................................


TUMOUR IMMUNOLOGY 5

Inhibitors of Apoptosis as Targets forSpontaneous T-cell Responses inCancer Patients: Potential UniversalAntigens in Therapeutic VaccinationsAgainst Cancer

M. H. Andersen,1 S. Reker,1 P. Kvistgaard,1

I. M. Svane,2 J. C. Becker3 & P. T. Straten1

1Tumor Immunology Group, Institute of Cancer Biology, Danish CancerSociety, 2Department of Oncology, State Hospital Herlev, and 3Departmentof Dermatology, University Hospital Wurzburg, Wurzburg, Germany.

Immunotherapy represents an attractive fourth-modalitytherapeutic approach, especially in the light of theshortcomings of conventional surgery, radiation andchemotherapies in the management of metastatic cancer.To this end, a large number of peptide antigens derivedfrom TAA have been applied in immunotherapeutictrials for the treatment of various malignancies, e.g. cancersof the breast, prostate and kidney, in addition tohaematological cancers. In some cases the responserates have been impressive and no adverse autoimmunityhave been observed. A major strategic difficulty associatedwith these trials relates to the choice of best-suitedpeptide antigens. The vast majority of the antigensdescribed thus far is not vital for survival and growth ofthe tumour cells, and immunoselection of antigen-lossvariants may therefore prove to be an additionalobstacle for the clinical applicability of most of theknown peptide epitopes. In this respect, the developmentof acquired antigen loss during immunotherapy hasbeen demonstrated in several cases. Obviously, thedevelopment of loss-variant tumour cells implies thatthese cells acquire a pronounced growth advantage andare left unaffected by further treatment. Ideally, targetantigens should be derived from proteins requiredfor survival and growth of tumour cells, as antigenswith these characteristics would not be inflicted by thedevelopment of loss-variant tumour cells. In this respect,several inhibitors of apoptosis proteins (IAPs) are univer-sally expressed among tumours and play an importantrole in tumour cell escape from apoptosis. We havecharacterized spontaneous T-cell reactivity against IAP-derived peptides in cancer patients. From the IAP survivin,we have characterized peptides restricted to the Class Imolecules HLA-A1, A2, A3, A11, B7 and B35. Further-more, we have demonstrated that survivin-specific T cellsinfiltrate metastatic lesions and that isolated survivin-specific CTLs are capable of killing HLA-matched tumourcells. Survivin-derived peptides are now in clinical trial,and continued work in our lab has demonstrated thatother IAPs are targets for spontaneous T-cell reactivity incancer patients.

TUMOUR IMMUNOLOGY 6

Functional and MolecularCharacterization of Myeloid SuppressorCells Expanded During LymphomaTumour Progression

S. Meerschaut, Y. Liu, G. H. G. Hassanzadeh,G. Raes, P. De Baetselier & J. Van GinderachterDepartment of Cellular and Molecular Immunology, Vrije UniversiteitBrussel, Flanders Interuniversity Institute for Biotechnology, Brussels,Belgium. E-mail: [email protected]

We previously reported that in mice with large progressingT-cell lymphoma tumours, dysfunctions in the antitumourCTL activity occur, associated with an accumulation ofsplenic arginase-producing myeloid suppressor cells(MSCs). In this study, we first demonstrate that both thepresence and the activation state of these MSC depends ontumour evolution. While in tumour regressors hardly anyarginase-producing MSC can be found, both the amountand the arginase activity of this population expands fromearly over late progressors. This gradual induction ofMSCs is paralleled by an increasing suppression of CTLactivity and Th1, but not Th2, cytokine production. Uponanalysing the molecular repertoire of MSC in vitro, wefound, besides arginase1, a well-established marker foralternatively activated myeloid cells or M2, a strong upre-gulation of FIZZ1 and Ym, two additional recently iden-tified markers for M2. Further evaluation of molecularmarkers by microarray analysis in MSC yielded genesinvolved in wound healing (e.g. coagulation factorXIIIa), anti-inflammation (e.g. selenoprotein P), immuno-modulation (e.g. PD-L2) and fat and sugar metabolism(e.g. leptin receptor). Of note, many of these genes areregulated by type 2 cytokines (IL-4, IL-13 and IL-10) andare therefore rather M2 associated. Overall, our data pro-vide new markers for MSC in cancer and further establishtheir M2 activation state.

TUMOUR IMMUNOLOGY 7

Clonally Expanded CD8þ T cells inAllogeneic Bone Marrow Transplantation

T. M. Kollgaard,1 S. Reker,1 S. L. Petersen,2

T. N. Masmas,2 L. L. Vindelov2 & P. T. Straten1

1Tumor Immunology Group, Institute of Cancer Biology, Danish CancerSociety, and 2The Lymphocyte Research Laboratory, Department ofHematology, Rigshospitalet, Copenhagen, Denmark. E-mail:[email protected]

Allogeneic bone marrow transplantation (BMT) is apotentially curative therapy for patients with haematologicmalignancies. Several lines of evidence demonstrate that

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donor T cells are involved in the antitumour effectsobserved after BMT. Thus, patients receiving T-cell-depleted BMT have a higher risk of leukaemia relapsecompared to patients receiving nonmanipulated BMT,and patients experiencing graft-versus-host disease(GVHD) have a lower risk of disease relapse than patientswho do not experience GVHD. Although the importanceof donor T cells for the curative action of BMT has beenestablished, the exact mechanisms and molecules involvedin this graft-versus-tumour effect remain largely unknown.In a recently initiated project, we have conducted a longi-tudinal study of T-cell clonotypes in patients whoreceived peripheral blood stem cell grafts after nonmyeloa-blative conditioning. Peripheral blood samples wereobtained sequentially after transplant, and the mononu-clear cells (MNCs) were isolated and cryopreserved. CD8þ

T cells were isolated from the MNCs by use of immuno-magnetic beads or FACS and analysed for the presence ofclonally expanded cells by T-cell receptor clonotype map-ping based on RT-PCR and denaturing gradient gel elec-trophoresis (DGGE). Using this gel-based methodology,clonally expanded T cells were monitored after transplantand compared to the clinical data of the patients. Thepreliminary results demonstrates the presence of clonallyexpanded CD8þ T cells at all time points analysed.Furthermore, a number of clonotypes persisted for morethan 6 months, and other clonotypes emerged during thisperiod. The appearance of newly emerged clonotypeswhich coincided with clinical GVHD could indicate arole for these T cells in the pathogenesis of GVHD.

TUMOUR IMMUNOLOGY 8

Preoperative Mannan-Binding LectinPathway and Prognosis in ColorectalCancer

H. Ytting,1 I. J. Christensen,1 S. Thiel,2

J. C. Jensenius2 & H. J. Nielsen1

1Department of Surgical Gastroenterology, Hvidovre University Hospital,Copenhagen, and 2Institute of Immunology and Microbiology, AarhusUniversity Hospital, Denmark. E-mail: [email protected]

Background: Deficiency of the mannan-binding lectin(MBL) pathway of innate immunity leads to increasedsusceptibility to infections. In patients with colorectalcancer, postoperative infection is associated with poorprognosis. The aim of the present study was to evaluate(1) the relation between the MBL pathway and postopera-tive infectious complications and survival of patientsresected for colorectal cancer and (2) the role of MBL asacute phase reactant compared to CRP.Methods: Preoperative MBL concentration, MBL/MBL-associated serine protease (MASP) activity and CRP were

determined in serum from 611 patients and 150 healthycontrols. The patients were observed for 8 years. Post-operative infections, recurrence and survival were recorded.Results: The MBL pathway components were increasedin the patients (P< 0.0001) compared to healthy controls.Low MBL levels were predictive of pneumonia (P¼ 0.01),and pneumonia (n¼ 87) was associated with poor survival(P¼ 0.003, HR¼ 1.5, 95% CI 1.1–1.9). MBL and MBL/MASP activity could not predict postoperative overallinfections. MBL showed no correlation (spearman’sr¼ 0.02, 95% CI �0.06–0.10) with CRP.Conclusions: Low preoperative MBL levels are predictiveof pneumonia, which is associated with poorer survival.MBL concentration and MBL/MASP activity was notpredictive of other postoperative infections or long-termprognosis. MBL apparently is not a surrogate measure ofCRP.

TUMOUR IMMUNOLOGY 9

Expression of Human Collectins inColorectal Carcinoma

M. Siassi, W. Hohenberger & R. CronerDepartment of Surgery, University Hospital of Erlangen, Erlangen,Germany. E-mail: [email protected]

Introduction: The human collectins, mannan-bindinglectin (MBL), surfactant protein-A (SP-A) and surfactant-protein-D (SP-D) play a central role in the innate immunesystem. Immunological responses to malignant transfor-mation of epithelial cells gained increasing interestrecently. A former study could demonstrate binding ofMBL to certain colorectal carcinoma (CRC) cell lines invitro. We therefore examined the expression of humancollectins in normal colon mucosa and in colorectalcarcinomas.Materials and methods: Colon samples from 20 CRCpatients and 10 normal mucosa samples were collectedimmediately after surgery. The tissue was microdissectedand RNA isolated (Qiagen, Rneasy-Kit). Gene expressionprofiles were analysed using Gene-chips (Affymetrix, HG-U133). We analysed the data for the expression of MBL,its associated serine proteases mannan-binding lectin-associated serine protease 1/2 (MASP 1/2), SP-A andSP-D. The signal intensity of the genes of interest wascompared using the Mann–Whitney U-test.Results: The expression of human collectins in normalhuman colon mucosa was generally low. Only the expres-sion of SP-A and MASP-2 reached the noise threshold of250 signals. These genes were significantly downregulatedin CRC specimens. The expression of the other proteinsshowed no difference in normal mucosa and CRC.Conclusion: As demonstrated before, the expression ofhuman collectins in normal colon was low in this

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study. Only SP-A showed a significant expression innormal mucosa which was downregulated in CRC. Asthe absolute signal level was below the noise threshold,these results have to be interpreted with caution andrequire confirmation by direct measurenment of theproteins. Our results suggest that there is no major rolefor the human collectins in colorectal cancer.

TUMOUR IMMUNOLOGY 10

Detection of Immune Responses inSentinel Nodes Draining Human UrinaryBladder Cancer

M. Karlsson,1 P. Marits,1 J. Baner,2 A. Sherif,3

M. Thorn,4 U. Landegren2 & O. Winqvist5

1Department of Internal Medicine, Uppsala University Hospital, 2Depart-ment of Genetics and Pathology, Rudbeck Laboratory, Uppsala University,3Department of Urology, Uppsala University Hospital, 4Department ofSurgery, South Stockholm General Hospital, Karolinska Institute, and5Department of Medicine, Section of Clinical Allergology and Immunology,Karolinska Hospital, Karolinska Institute, Stockholm, Sweden. E-mail:[email protected]

Being the first lymph node to receive drainage from thetumour area, the sentinel node offers a unique possibility

to obtain tumour-reactive lymphocytes. We investigatedantitumour immune responses in sentinel nodes frompatients with bladder cancer, by assaying tumour-specificproliferation and TCR Vb repertoires. During tumoursurgery, sentinel lymph nodes were identified by peri-tumoural injection of blue dye. Fresh specimens oftumour, sentinel and nonsentinel lymph nodes wereobtained, and single-cell suspensions were prepared. Cellswere assayed for reactivity against autologous tumourextract in [3H]-thymidine incorporation assays and char-acterized by flow cytometry. Parallel analyses of the expres-sion of Vb gene families were performed with padlockprobes, linear oligonucleotides which upon target recogni-tion can be converted to circular molecules by a ligase.Probes were reacted with cDNA prepared from magneti-cally separated CD4þ cells, and the TCR repertoire wasdetermined by hybridizing the products to oligonucleotidemicroarrays. Dose-dependent proliferation in response totumour extract could be detected in sentinel lymph nodes.Common clonal expansions were detected among tumour-infiltrating lymphocytes and in sentinel lymph nodes.Nonsentinel lymph nodes displayed a divergent TCR Vbrepertoire. These results indicate an ongoing immuneresponse against tumour antigens in sentinel nodes, drain-ing urinary bladder cancer. Identification of sentinellymph nodes makes it possible to obtain tumour-reactivelymphocytes for use in adoptive immunotherapy.

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Detection of Immune Responses in Sentinel Nodes Draining Human Urinary Bladder Cancer

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